Gas-filled structures and related compositions, methods and systems to image a target site

ABSTRACT

Gas vesicles, protein variants and related compositions methods and systems for singleplexed and/or multiplexed ultrasound imaging of a target site in which a gas vesicle provides contrast for the imaging which is modifiable by application of a selectable acoustic collapse pressure value of the gas vesicle.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 62/344,498, entitled “Genetically Engineered Gas-FilledNanostructures” filed on Jun. 2, 2016, which is incorporated herein byreference in its entirety.

STATEMENT OF INTEREST

This invention was made with government support under Grant No.W911NF-14-1-0111 awarded by the Army and under Grant No. EB018975awarded by the National Institute of Health. The government has certainrights in the invention.”

FIELD

The present disclosure relates to gas-filled structures for use inimaging technologies, and related compositions methods and systems toimage a target site with particular reference to imaging performed byultrasound.

BACKGROUND

Ultrasound is among the most widely used biomedical imaging modalitiesdue to its superior spatiotemporal resolution, safety, cost and ease ofuse compared to other techniques.

In addition to visualizing anatomy and physiology, ultrasound can takeadvantage of contrast agents to more specifically image blood flow,discern the location of certain molecular targets, and resolvestructures beyond its normal wavelength limit via super-localization.

Challenges remain for identifying and developing methods andbiocompatible nanoscale contrast agents for ultrasound detection of atarget site obtained with high sensitivity and resolution.

SUMMARY

Provided herein are gas vesicles protein structures (GVs) with tunableacoustic properties and related compositions methods, and systems whichcan be used in several embodiments to perform harmonic, multiplexed andmultimodal ultrasound imaging, as well as cell-specific moleculartargeting.

According to a first aspect, a method to provide an ultrasound imagingof a target site contrasted with a gas vesicle protein structure (GVPS)is described. In the method, the GVPS has a selectable acoustic collapsepressure value derived from an acoustic collapse pressure profile of theGVPS type and a hydrostatic collapse pressure profile, and a midpoint ofthe acoustic collapse pressure profile higher than a midpoint of thehydrostatic collapse pressure profile. The method comprises: collapsingthe GVPS type by applying collapsing ultrasound to the target site, thecollapsing ultrasound applied at a collapsing ultrasound pressuregreater than the selectable acoustic collapse pressure value. The methodfurther comprises imaging the target site by applying imaging ultrasoundto the target site, the imaging ultrasound applied at a first imagingultrasound pressure selected to provide an uncontrasted image of thetarget site. In some embodiments, the collapsing ultrasound pressure ishigher than the midpoint of the hydrostatic collapse pressure profile.

According to a second aspect, an ultrasound imaging method and system tobe used on a target site contrasted with a first gas vesicle proteinstructure (GVPS) type are described. In the method the GVPS typeexhibits a first acoustic collapse pressure profile and a firstselectable acoustic collapse pressure value and a second GVPS typeexhibiting a second acoustic collapse pressure profile and a secondselectable acoustic collapse pressure value. In the method, each GVPStype exhibiting a different acoustic collapse pressure profile definedas a collapse function from which a collapse amount can be determinedand a different selectable acoustic collapse pressure value from theircorresponding acoustic collapse pressure profile. The method comprisesselectively collapsing the first GVPS type by applying collapsingultrasound to the target site, the collapsing ultrasound applied at afirst acoustic collapse pressure value equal to or higher than the firstselectable acoustic collapse pressure value and lower than the secondselectable acoustic collapse pressure value. The method furthercomprises imaging the target site containing second, uncollapsed, GVPStype by applying imaging ultrasound to the target site, the imagingultrasound applied at a pressure value lower than the acoustic collapsepressure value of the second gas vesicle structure type. The systemcomprises the first GVPS and the second GVPS in a combination forsimultaneous sequential use in the ultrasound imaging method hereindescribed.

According to a third aspect, an ultrasound imaging method and system tobe used on a target site contrasted with a plurality of gas vesicleprotein structure (GVPS) types are described. In the method, each GVPStype exhibits i) an acoustic collapse pressure profile defined as acollapse function from which a collapse amount can be determined, andii) a selectable acoustic collapse pressure value, selectable acousticcollapse pressure values going from a lowest acoustic collapse pressurevalue to a highest acoustic collapse pressure value. the methodcomprises selectively collapsing GVPS type to a collapse amount higherthan a collapse amount of each remaining GVPS type by applyingcollapsing ultrasound to the target site, the collapsing ultrasoundapplied at a pressure value equal to or higher than the selectableacoustic collapse pressure value of the GVPS type being collapsed andlower than an acoustic collapse pressure value of said each remainingGVPS type or types. The method further comprises imaging the target sitecontaining the remaining GVPS type or types by applying imagingultrasound to the target site, the imaging ultrasound applied at apressure value lower than a lowest acoustic collapse pressure value ofsaid each remaining GVPS type or types. The method also comprisesrepeating the collapsing and the imaging until all GVPS types arecollapsed, thus providing a sequence of visible images of the targetsite, the sequence being indicative of image-by-image decreasingremaining GVPS types. The system comprises a plurality of gas vesicleprotein structure (GVPS) types in a combination for simultaneoussequential use in the ultrasound imaging method herein described.

According to a fourth aspect, a method and system to tune acousticproperties of a gas vesicle protein structure (GVPS) are described. Thegas vesicle protein structure (GVPS) is formed by a gas enclosed by aprotein layer formed by a GvpC protein attached to other gas vesicleproteins to form the protein layer configured to be permeable to gas butnot liquid. The GVPS has a selectable acoustic collapse pressure valueaP₀ derived from an acoustic collapse pressure profile of the GVPS and ahydrostatic collapse pressure profile, and a midpoint of the acousticcollapse pressure profile higher than a midpoint of the hydrostaticcollapse pressure profile. The method comprises engineering the GVPS byreplacing a GvpC protein of the GVPS with: subsaturated concentrationsof the GvpC protein and/or saturated or subsaturated concentrations of agenetically modified GvpC protein. In the method, the engineering isperformed to obtain a variant of the GvpC protein with an acousticcollapse pressure aP1 lower than the aP₀.

According to a fifth aspect, a gas vesicle protein structure variant isdescribed. The gas vesicle protein structure comprising a variant GvpCprotein herein described.

According to a sixth aspect, a GvpC protein variant is describedobtainable from a base GvpC protein having repetitions of a repeatregion flanked by an N-terminal region, and a C-terminal region, bydeleting at least one of the N-terminal region and C-terminal region;deleting 3 or more repeated region; deleting at least one repeatedregion immediately following the N-terminus, and/or adding a peptide tothe C terminal or N terminal region.

According to a seventh aspect, a GvpC protein variant of a base GvpCprotein having repetitions of a repeat region, flanked by an N-terminalregion and a C-terminal region, is described. The GvpC variant comprisesone or more repeat regions with a sub-sequence within at least onerepeat region substituted with another amino sequence having a sequencesimilarity lower than 50% with respect to the sub-sequence within thebase GvpC sequence.

According to an eight aspect, a composition is described comprising oneor more GVPS, and/or one or more GvpC variant herein described and asuitable vehicle. In some embodiments, wherein the composition comprisesone or more GVPS, the composition is a contrast agent for ultrasoundimaging.

The gas vesicle protein structures and related variants, compositionsmethods and systems as well as GvpC variants herein described can beused in several embodiments in connection with ultrasound imaging ofbiological target site with particular reference to imaging of internalbody structures of an individual such as tendons, muscles, joints,vessels and internal organs.

The gas vesicle protein structures and related variants, compositionsmethods and systems as well as GvpC variants herein described can beused in several embodiments to provide ultrasound imaging with enhancedharmonic responses, biodistribution, multiplexing, multimodal detectionand/or molecular targeting to help ultrasound fulfill its potential as ahigh performance modality for molecular imaging.

The gas vesicle protein structures and related variants, compositionsmethods and systems as well as GvpC variants herein described can beused in several embodiments to track moving target sites such as cellsor other structures within the body of an individual or otherenvironments

The gas vesicle protein structures and related variants, compositionsmethods and systems as well as GvpC variants herein described can beused in connection with various applications wherein ultrasound imagingof a target site is desired. For example, The gas vesicle proteinstructures and related variants, compositions methods and systems aswell as GvpC variants herein described can be used to spatially and/ortemporally control the contrast of the imaging of a biological targetsite and in particular internal body structure or molecular compositionor cellular composition and activity of tissues of an individual inmedical applications, as well diagnostics applications. Additionalexemplary applications include uses of gas vesicle protein structuresand related variants, compositions methods and systems as well as GvpCvariants herein described in several fields including basic biologyresearch, applied biology, bio-engineering, bio-energy, medicalresearch, medical diagnostics, therapeutics, and in additional fieldsidentifiable by a skilled person upon reading of the present disclosure.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description and theexamples, serve to explain the principles and implementations of thedisclosure.

FIG. 1 shows a rendition of engineered GVs illustrating GvpA as the mainbuilding block of GVs. GvpA is a structural protein that assemblesthrough repeated unites to make up the bulk of GVs. GvpC is a scaffoldprotein with 5 repeat units that assemble on the outer shell of GVs.GvpC can be engineered to tune the mechanical and acoustic properties ofGVs as well as act as a handle for appending moieties on to.

FIG. 2 shows schematics showing GVs as a molecular engineering platformfor acoustic protein structures. Panel a shows an exemplary TransmissionElectron Microscopy (TEM) image of a single Ana GV. Panel b shows aschematic illustration of Ana GV, and the gene cluster encoding GvpA,GvpC and several other essential proteins. Panel c shows GvpA and GvpCare the two major structural constituents of GVs, with GvpA ribs (lightgray) forming the primary GV shell and the outer scaffold protein GvpC(dark gray) conferring structural integrity. Each GvpC molecule has five33-amino acid repeats flanked by N- and C-terminal regions. Panel dshows a schematic of an exemplary paradigm for modular geneticengineering of Ana GVs. Native gas vesicles are treated with 6M urea toproduce stripped Ana GVs without native GvpC. Genetically engineeredGvpC is recombinantly expressed in Escherichia coli and added to thestripped Ana GVs during dialysis to create engineered GVs with amodified GvpC layer. Panel e shows a schematic of examples of how GvpCengineering can be used to modulate the properties of acoustic GVstructures including their harmonic response, collapse pressure, surfacecharge, targeting specificity and fluorescence.

FIG. 3 shows an exemplary SDS-PAGE analysis confirming the completeremoval of GvpC from native Ana GVs (lane 13) and the re-addition ofengineered proteins (lane 14-15). Quantification of re-added GvpC onurea-stripped Ana GVs was done by comparison against a standard curve(200-1000 ng) of the pure proteins (lanes 2-6 for WT-GvpC and lanes 7-11for ΔN&C-GvpC). The number of re-added GvpC molecules was determined tobe ˜1980 per GV for GvpC_(WT) and ˜877 per GV for ΔN&C respectively.

FIG. 4 shows examples of GvpC engineering that enables tuning of GVcollapse pressure for acoustic multiplexing. Panel a shows a schematicillustration of three exemplary engineered GV variants used for acousticmultiplexing. ΔGvpC is shown with no gvpC (no diagonal lines), ΔN&C isshown with light grey diagonal lines representing ΔN&C gvpC mutant, andGvpC_(WT) is shown with dark grey diagonal lines (WT gvpC). AccompanyingTEM images (to the right of respective illustrated GV variants) show theconservation of GV shape among the three variants (scale bars are 200nm). Panel b shows graphed results of exemplary optical density (O.D.)measurements of engineered Ana GVs as a function of hydrostatic pressure(N=7 independent preparations, error bars are SEM). Panel c showsgraphed exemplary results of acoustic collapse curves for the GVvariants showing normalized ultrasound signal intensity as a function ofincreasing peak positive pressure from 290 kPa to 1.23 MPa (N=3independent trials, error bars are SEM). The data was fitted with aBoltzmann sigmoid function (parameters provided in Table 7), thederivatives of which with respect to pressure are plotted in Panel d.Panel e shows a schematic illustration of acoustic spectral unmixing,showing serial collapse of the exemplary GV variants based on theircritical collapse pressure and indicating the pressures used in Panels fand g. Panel f shows exemplary ultrasound images of an agarose phantomcontaining wells with ΔGvpC, ΔN&C, GvpC_(WT) and a mixture of the threevariants (all GVs at final OD 1.0 in PBS), acquired at 6.25 MHz. I₀;before collapse I₁: after collapse at 630 kPa I2: after collapse at 790kPa 13: after collapse at 1230 kPa. Panel g shows exemplary spectrallyunmixed images processed from the raw ultrasound data in (Panel f). Thebottom row of images in Panel g shows an overlay of the three unmixedchannels C₁, C₂, and C₃. The color version of FIG. 4 can be found in theLakshmanan reference [1], which is incorporated by reference in itsentirety.

FIG. 5 shows a graph reporting exemplary results of midpoint of collapse(hydrostatic) plotted as a function of re-added GvpC concentration forthe ΔN&C variant. The midpoint of collapse was determined by fitting theraw data with a Boltzmann sigmoid function of the form ƒ(p)=(1+e^((p-p)^(c) ^()/Δp))⁻¹ with p_(c) representing the average midpoint ofcollapse. Fit parameters and R² values for each of the GV variants areprovided in the table below the graph. The saturation curve was plottedby fitting the data to a bimolecular binding function of the formf(x)=C₁*x/(K_(d)+x)+C₂.

FIG. 6 shows a graph reporting exemplary results of midpoint of collapse(hydrostatic) plotted as a function of re-added GvpC concentration forthe GvpC_(WT) variant. The midpoint of collapse was determined byfitting the raw data with a Boltzmann sigmoid function of the formƒ(p)=(1+e^((p-p) ^(c) ^()/Δp))⁻¹ with p_(c) representing the averagemidpoint of collapse. Fit parameters and R² values for each of the GVvariants are provided in the table below the graph. The saturation curvewas plotted by fitting the data to a bimolecular binding function of theform f(x)=C₁*x/(K_(d)+x)+C₂.

FIG. 7 shows an exemplary matrix of coefficients used for generatingspectrally unmixed images shown in FIG. 4 Panel g from the pixel-wiseultrasound signal intensities in FIG. 4 Panel f (I), before and afterexposing the GV samples to three sequentially increasing acousticpressures (P_(i)). Δ represents the measured differential signals withΔ_(i)=I(P_(i-1))−I(P_(i)), while α is the matrix containing the acousticcollapse spectrum for each GV variant (α_(i,j)). C represents thecontribution of each GV variant to the observed signal, with C₁calculated by the matrix operation: C=α⁻¹Δ.

FIG. 8 shows exemplary results showing that GV engineering enablesmodulation of harmonic signals in vitro. Panel a shows a graph reportingexemplary power spectra of signal backscattered from ΔGvpC (light grey)and GvpC_(WT) (dark grey) variants in an agarose phantom in response to4.46 MHz pulses. Panel b shows exemplary fundamental and Panel c secondharmonic ultrasound images of ΔGvpC and GvpC_(WT) GVs acquired with 4.46MHz transmission and band-pass filtered around 4.46 and 8.92 MHzrespectively. Images are shown before and after collapse using a highpower burst from the transducer to collapse the GVs. Scale bars are 1mm. Panel d-e show graphs reporting exemplary mean fundamental (in Paneld) and harmonic (in Panel e) signals from ΔGvpC and GvpC_(WT) variantsafter filtering at the indicated frequencies (N=7 independentmeasurements, error bars are SEM). Data in all panels comes from GVsprepared at OD 2.5 in PBS and loaded into 1% agarose phantoms.

FIG. 9 shows exemplary images and graphed results showing that GVengineering enables modulation of harmonic signals in vivo. Panel ashows a schematic depiction of intravenous GV injection and in vivoultrasound imaging during passage through the inferior vena cava (IVC).Panel b shows exemplary fundamental and second harmonic ultrasoundimages taken at 4.46 MHz transmission frequency and band-pass filteredreceive around 4.46 and 8.92 MHz respectively. Engineered Ana GVs at OD23.5 in PBS were used for injections. The IVC ROI used for subsequentanalysis is circled with a dashed line. The white arrow points to theincreased harmonic signal observed in the IVC for the ΔGvpC variant.Panels c-d show graphs reporting exemplary results of time course of themean (Panel c) fundamental and (Panel d) harmonic acoustic signal in theIVC before, during and after steady infusion, with shaded regionsrepresenting SEM (N=6 mice). Panel e shows a histogram reportingexemplary area under the curve (AUC) of average fundamental and harmoniccontrast in the IVC after ΔGvpC and GvpC_(WT) GV injections (N=6, errorbars are SEM).

FIG. 10 shows examples of genetic engineering of GV surface properties,cellular targeting and multimodal imaging. Panel a shows a diagram ofgvpC genetic fusions used to engineer novel GV properties and functions.Panel b shows exemplary graphed results of Zeta potential measurementsof engineered GVs having GvpC fused to LRP and wild-type GvpC (N=4,error bars are SEM). Panel c shows exemplary confocal fluorescenceimages showing RGD-functionalized, RDG-functionalized and wild-typeAlexa Fluor-488 fluorescently labeled (white) GVs after 24 hr incubationwith U87 glioblastoma cells (DAPI-stained nuclei, light gray). Scalebars are 50 μm. Panel d shows exemplary graphed results of mean GVfluorescence measured for each condition in Panel c (N=3, error bars areSEM). Panel e shows exemplary confocal fluorescence images of RAW 264.7macrophages (DAPI-stained nuclei, light gray) incubated for 30 min withfluorescently labeled GVs (white) displaying GvpC fused to mCD47, R8 orwild-type GvpC. Scale bars are 50 μm. Panel f shows exemplary graphedresults of mean GV fluorescence measured for each condition in Panel e(N=3, error bars are SEM). Panel g shows in the Top row: exemplaryultrasound images of engineered and SpyCatcher-mNeonGreen (SC-mNG)reacted GVs at OD 2.5 in PBS, acquired using a 19 MHz transmission pulsein fundamental mode. Scale bars are 1 mm. The bottom row of Panel gshows exemplary fluorescence images of the agarose phantoms before andafter acoustic collapse. Panel h shows exemplary graphed results of meanultrasound and fluorescence signals from the GV samples tested in Panelg (N≥4, error bars are SEM).

FIG. 11 shows a Clustal Omega sequence alignment of exemplarygenetically engineered variant GvpC proteins described herein. Inparticular, FIG. 11 shows the amino acid sequence of the followingvariants: ΔN&C-CERY1 (SEQ ID NO:35), Δnterm (SEQ ID NO:36), N-rep3-C(SEQID NO:37), SR3CERY1 (SEQ ID NO:38), WTCERY1 (SEQ ID NO:39), N-rep1-C(SEQID NO:40), SR10ERY1 (SEQ ID NO:41), ΔN&C (SEQ ID NO:42),N-rep2endto3mid-C(SEQ ID NO:43), N-His-GvpC (SEQ ID NO:44), ΔCterm (SEQID NO:45), GvpCWT-ACPP (SEQ ID NO:46), GvpCWT-hPRM (SEQ ID NO:47),GvpCWT-LRP (SEQ ID NO:48), GvpCWT-mCD47 (SEQ ID NO:49), GvpCWT-R8 (SEQID NO:50), GvpCWT-RGD (SEQ ID NO:51), GvpCWT-RDG (SEQ ID NO:52), GvpCWT(SEQ ID NO:53), GvpC-SpyTag (SEQ ID NO:54), N-rep1to3-C(SEQ ID NO:55),N-rep1to2-C(SEQ ID NO:56), and N-rep1to4-C(SEQ ID NO:57).

FIG. 12 shows a graph reporting exemplary optical density measurementsof engineered Ana GVs as a function of hydrostatic pressure. The datawas fitted with the Boltzmann sigmoid function ƒ(p)=(1+e^((p-p) ^(c)^()/Δp))⁻¹ and the table provides the midpoint of collapse as well asother fit parameters and R² values. The data show that the collapseprofile is unaltered even after reacting the ST-GVs with SC-mNGfluorescent protein.

FIG. 13 shows an exemplary SDS-PAGE quantification of SpyTagfunctionalities on the surface of engineered Ana GVs. Comparison ofST-Ana GVs (lane 10) against a standard curve comprising GvpC-STconcentrations ranging from 100-1000 ng (lanes 2-8) shows that eachmodified GV has ˜1000 SpyTag functionalities. Stripped Ana GVs used forGvpC-ST re-addition (lane 9) have negligible amount of native GvpC.

FIG. 14 shows SDS-PAGE analysis confirms SpyTag-SpyCatcher bondformation (dashed box around band in lane 5) upon a one-hour incubationof ST-GVs having an outer layer of GvpC-SpyTag (dashed boxes aroundbands in lanes 2 and 4) with SpyCatcher-mNeonGreen (dashed box aroundband in lane 3). Incubation of Ana GVs containing an outer layer ofWT-GvpC (dashed box around band in lane 6) with SC-mNG, followed bybuoyancy purification to remove unreacted fluorescent molecules resultsin GVs that are not fluorescent as shown in the left image in bottom rowof images in FIG. 10 Panel g. This also highlights the specificity ofthe SpyTag-SpyCatcher reaction and confirms that all the unreactedfluorescent molecules are completely removed during buoyancypurification.

FIG. 15 shows an illustration of an exemplary collapsometry setup usedfor determining the hydrostatic critical collapse pressure of GVs,wherein 1501 shows a nitrogen cylinder, 1502 shows a pressurecontroller, 1503 shows an optical density detector set an 500 nm, 1504shows a cuvette containing GV solution, and 1505 shows a lamp forillumination of the cuvette.

FIG. 16 shows amino acid GvpC sequences from 5 different organisms withthe tandem repeat regions (Rep) within each gvpC protein aligned,preceded by the N-terminal region (N-term) and followed by theC-terminal region (C-term). Sequences and tandem repeats were obtainedfrom Uniprot. Panel a shows Halobacterium salinarum, (SEQ ID NO:4),Panel b shows Anabaena floc-aquae, (SEQ ID NO:2), Panel c showsHalobacterium mediterranei, (SEQ ID NO:6), Panel d Microchaetediplosiphon, (SEQ ID NO:8), and Panel e shows Nostoc sp., (SEQ IDNO:10).

FIG. 17 shows a table summarizing results of experiments on GVs preparedusing exemplary gvpC variants described herein. For each variant, themolar concentration ratio of gvpC:gvpA used for dialysis assembly of GVsis shown (concentration). The midpoint of hydrostatic collapse pressure(pressure at which half the GV population collapse) is shown for eachpreparation of GVs indicated. For each preparation, it is also indicatedwhether the gvpC variant has a His-tag, and the location of the His-tagon the gvpC protein (C-terminus or N-terminus).

FIG. 18 shows GvpC amino acid sequence from Anabaena floc-aquae, (SEQ IDNO:2), with the tandem repeat regions (Rep) aligned, preceded by theN-terminal region (N-term) and followed by the C-terminal region(C-term). Underlined residues indicate sites of trypsin cleavage, andresidues in bold indicate regions of the protein that remain bound togvpA following a tryptic digest.

FIG. 19 shows examples of GvpC engineering that enables tuning of GVcollapse pressure for acoustic multiplexing. Panel a shows graphedresults of exemplary optical density (O.D.) measurements of engineeredAna GVs as a function of hydrostatic pressure. GVs prepared with Rep1,Rep2to3, or Rep3 variants of gvpC were prepared 7.5:25 molar ratio ofgvpC:gvpA and for the GvpC_(WT) variant a gvpC:gvpA molar ratio of 2:25was used. The data was fitted with a Boltzmann sigmoid function. Panel bshows a table summarizing the midpoint of collapse (kPa) of eachpreparation shown in Panel a.

FIG. 20 shows an exemplary SDS-PAGE analysis confirms that the indicatedgvpC variants bound to gvpA of intact GVs (shown in boxes around bands).The results indicate that truncated single repeat variants bind to GVsbut do not strengthen the GV wall to the same extent. All samples werebetween OD₅₀₀: 5.5 to 6.5 before 1:1 dilution with SDS-loading buffer.

FIG. 21 shows examples of GvpC engineering that enables tuning of GVcollapse pressure for acoustic multiplexing. Panel a shows graphedresults of exemplary optical density (O.D.) measurements of wild typeAna GVs (GV_(WT)) and engineered Ana GVs as a function of hydrostaticpressure. GVs were prepared with the indicated gvpC variants (gvpC:gvpAequimolar). The data was fitted with a Boltzmann sigmoid function. Panelb shows a table summarizing the midpoint of collapse (kPa) of eachpreparation shown in Panel a.

FIG. 22 summarizes features of exemplary GV forming microorganisms [2].

FIG. 23 summarizes predicted products of gvp genes from Halobacteriumhalobium, Halobacterium salinarium, and Haloferax meditteranei [3].

FIG. 24 shows a chart illustrating an exemplary diagram showing acousticcollapse pressure midpoints and hydrostatic collapse pressure midpointsfor exemplary Ana GVs.

FIG. 25 shows a chart illustrating an exemplary diagram showing acousticcollapse pressure midpoints and hydrostatic collapse pressure midpointsfor exemplary Halo GVs.

FIG. 26 shows exemplary sequence alignment of GvpC repeat regions fromdifferent organisms. Panel a shows Halobacterium salinarum, r7-halsa(SEQ ID NO:58), r6-halsa (SEQ ID NO:59), r5-halsa (SEQ ID NO:60),r2-halsa (SEQ ID NO:61), r3-halsa (SEQ ID NO:62), r1-halsa (SEQ IDNO:63), r4-halsa (SEQ ID NO:64), Panel b shows Haloferax mediterranei,r7-halmed (SEQ ID NO:65), r6-halmed (SEQ ID NO:66), r1-halmed (SEQ IDNO:67), r3-halmed (SEQ ID NO:68), r4-halmed (SEQ ID NO:69), r2-halmed(SEQ ID NO:70), r5-halmed (SEQ ID NO:71), Panel c shows Anabaenafloc-aquae, r1-ana (SEQ ID NO:72), r5-ana (SEQ ID NO:73), r4-ana (SEQ IDNO:74), r2-ana (SEQ ID NO:75), r3-ana (SEQ ID NO:76), Panel dMicrochaete diplosiphon, r1-microchaete (SEQ ID NO:77), r3-microchaete(SEQ ID NO:78), r4-microchaete (SEQ ID NO:79), Panel e shows Nostoc sp.r1-Nostoc (SEQ ID NO:80), r2-Nostoc (SEQ ID NO:81), r3-Nostoc (SEQ IDNO:82), Panel f shows Microcystise aeruginosa, r1-microcystis (SEQ IDNO:83), r2-microcystis (SEQ ID NO:84), r3-microcystis (SEQ ID NO:85),r4-microcystis (SEQ ID NO:86), r5-microcystis (SEQ ID NO:87), andr6-microcystis (SEQ ID NO:88), Panel g shows a sequence alignment of theconsensus sequences from Halobacterium salinarum and Haloferaxmediterranei, Halmed-consensus (SEQ ID NO:89), and Halsa-consensus (SEQID NO:90), Panel h shows a sequence alignment of the consensus sequencesfrom Anabaena floc-aquae, Microchaete diplosiphon and Nostoc sp.,Ana-consensus (SEQ ID NO:91), Microchaete-consensus (SEQ ID NO:92), andNostoc-consensus (SEQ ID NO:93).

FIG. 27 shows an exemplary schematic illustration of a multiplexingultrasound imaging method. N different GV types are each assigned anindex, n, from 1 to N according to their midpoint acoustic collapsepressure, in ascending order. P_(image) is the imaging pressure, chosento be below the lowest initial collapse pressure. P_(MIAP(n)) is theMIAP relating the n'th GV type and (n+1)'th GV type. P_(final) is apressure above the highest complete collapse pressure within the GVtype. This procedure results in N+1 images, which are then used toreconstruct the relative abundance of each GV type via spectralunmixing. The step in the bracket can be skipped if N=1.

DETAILED DESCRIPTION

Provided herein are gas-filled protein structures, also referred to as“gas vesicles” (GVs), and related compositions methods and systems foruse in ultrasound imaging particularly in contrast enhanced ultrasoundimaging.

The term “contrast enhanced imaging” or “imaging”, as herein indicates avisualization of a target site performed with the aid of a contrastagent administered to the target site to improve the visibility ofstructures or fluids by devices process and techniques suitable toprovide a visual representation of a target site. Accordingly contrastagent is a substance that enhances the contrast of structures or fluidswithin the target site, producing a higher contrast image forevaluation.

The term “ultrasound imaging” or ultrasound scanning” or “sonography” asused herein indicate imaging performed with techniques based on theapplication of ultrasound. Ultrasound refers to sound with frequencieshigher than the audible limits of human beings, typically over 20 kHz.Ultrasound devices typically can range up to the gigahertz range offrequencies, with most medical ultrasound devices operating in the 1 to18 MHz range. The amplitude of the waves relates to the intensity of theultrasound, which in turn relates to the pressure created by theultrasound waves. Applying ultrasound can be accomplished, for example,by sending strong, short electrical pulses to a piezoelectric transducerdirected at the target. Ultrasound can be applied as a continuous wave,or as wave pulses as will be understood by a skilled person.

Accordingly, the wording “ultrasound imaging” as used herein refers inparticular to the use of high frequency sound waves, typically broadbandwaves in the megahertz range, to image structures in the body. The imagecan be up to 3D with ultrasound. In particular, ultrasound imagingtypically involves the use of a small transducer (probe) transmittinghigh-frequency sound waves to a target site and collecting the soundsthat bounce back from the target site to provide the collected sound toa computer using sound waves to create an image of the target site.Ultrasound imaging allows detection of the function of moving structuresin real-time. Ultrasound imaging works on the principle that differentstructures/fluids in the target site will attenuate and return sounddifferently depending on their composition. A contrast agent sometimesused with ultrasound imaging are microbubbles created by an agitatedsaline solution, which works due to the drop in density at the interfacebetween the gas in the bubbles and the surrounding fluid, which createsa strong ultrasound reflection. Ultrasound imaging can be performed withconventional ultrasound techniques and devices displaying 2D images aswell as three-dimensional (3-D) ultrasound that formats the sound wavedata into 3-D images. In addition to 3D ultrasound imaging, ultrasoundimaging also encompasses Doppler ultrasound imaging, which uses theDoppler Effect to measure and visualize movement, such as blood flowrates. Types of Doppler imaging includes continuous wave Doppler, wherea continuous sinusoidal wave is used; pulsed wave Doppler, which usespulsed waves transmitted at a constant repetition frequency, and colorflow imaging, which uses the phase shift between pulses to determinevelocity information which is given a false color (such as red=flowtowards viewer and blue=flow away from viewer) superimposed on agrey-scale anatomical image. Ultrasound imaging can use linear ornon-linear propagation depending on the signal level. Harmonic andharmonic transient ultrasound response imaging can be used for increasedaxial resolution, as harmonic waves are generated from non-lineardistortions of the acoustic signal as the ultrasound waves insonatetissues in the body.

Other ultrasound techniques and devices suitable to image a target siteusing ultrasound would be understood by a skilled person.

The term “target site” as used herein indicates an environmentcomprising one or more targets intended as a combination of structuresand fluids to be contrasted, such as cells. In particular the term“target site” refers to biological environments such as cells, tissues,organs in vitro in vivo or ex vivo that contain at least one target. Atarget is a portion of the target site to be contrasted against thebackground (e.g. surrounding matter) of the target site. Accordingly, atarget can include any molecule, cell, tissue, body part, body cavity,organ system, whole organisms, collection of any number of organismswithin any suitable environment in vitro, in vivo or ex vivo as will beunderstood by a skilled person. Exemplary target sites includecollections of microorganisms, including, bacteria or archaea in asolution in vitro, as well as cells grown in an in vitro culture,including, primary mammalian, cells, immortalized cell lines, tumorcells, stem cells, and the like. Additional exemplary target sitesinclude tissues and organs in an ex vivo colture and tissue, organs, ororgans systems in a subject, for example, lungs, brain, kidney, liver,heart, the central nervous system, the peripheral nervous system, thegastrointestinal system, the circulatory system, the immune system, theskeletal system, the sensory system, within a body of an individual andadditional environments identifiable by a skilled person. The term“individual” or “subject” or “patient” as used herein in the context ofimaging includes a single plant or animal and in particular higherplants or animals and in particular vertebrates such as mammals and moreparticularly human beings. Types of ultrasound imaging of biologicaltarget sites include abdominal ultrasound, vascular ultrasound,obstetrical ultrasound, hysterosonography, pelvic ultrasound, renalultrasound, thyroid ultrasound, testicular ultrasound, and pediatricultrasound as well as additional ultrasound imaging as would beunderstood by a skilled person.

In embodiments herein described the ultrasound imaging of target site ifperformed in connection with the administration to the target site ofgas vesicle protein structures.

The wordings “gas vesicles protein structure” or “GV”, “GVP” or “GasVesicles” as used herein refer to a gas-filled protein structureintracellularly expressed by certain bacteria or archea as a mechanismto regulate cellular buoyancy in aqueous environments [3]. Inparticular, gas vesicles are protein structures natively expressedalmost exclusively in microorganisms from aquatic habitats, to providebuoyancy by lowering the density of the cells [3]. GVs have been foundin over 150 species of prokaryotes, comprising cyanobacteria andbacteria other than cyanobacteria [4, 5], from at least 5 of the 11phyla of bacteria and 2 of the phyla of archaea described by Woese(1987) [6]. Exemplary microorganisms expressing or carrying gas vesicleprotein structure include cyanobacteria such as Microcystis aeruginosa,Aphanizomenon flos aquae Oscillatoria agardhii, Anabaena, Microchaetediplosiphon and Nostoc; phototropic bacteria such as Amoebobacter, Thiodiclyon, Pelodiclyon, and Ancalochloris; non phototropic bacteriasuch as Microcyclus aquaticus; Gram-positive bacteria such as Bacillusmegalerium; Gram-negative bacteria such as Serratia; and archaea such asHaloferax mediterranei, Methanosarcina barkeri, Halobacteria salinariumas well as additional microorganisms identifiable by a skilled person.

In particular, a GV in the sense of the disclosure is a structureintracellularly expressed by bacteria or archea forming a hollowstructure wherein a gas is enclosed by a protein shell, which is a shellsubstantially made of protein (up at least 95% protein). In gas vesiclesin the sense of the disclosure, the protein shell is formed by aplurality of proteins herein also indicated as Gyp proteins or Gvps,which are expressed by the bacteria or archea and form in the bacteriaor archea cytoplasm a gas permeable and liquid impermeable protein shellconfiguration encircling gas. Accordingly, a protein shell of a GV ispermeable to gas but not to surrounding liquid such as water. Inparticular, GVs' protein shells exclude water but permit gas to freelydiffuse in and out from the surrounding media [7] making them physicallystable despite their usual nanometer size, unlike microbubbles, whichtrap pre-loaded gas in an unstable configuration.

GV structures are typically nanostructures with widths and lengths ofnanometer dimensions (in particular with widths of 45-250 nm and lengthsof 100-800 nm) but can have lengths up to 2 μm as will be understood bya skilled person. In certain embodiments, the gas vesicles proteinstructure have average dimensions of 1000 nm or less, such as 900 nm orless, including 800 nm or less, or 700 nm or less, or 600 nm or less, or500 nm or less, or 400 nm or less, or 300 nm or less, or 250 nm or less,or 200 nm or less, or 150 nm or less, or 100 nm or less, or 75 nm orless, or 50 nm or less, or 25 nm or less, or 10 nm or less. For example,the average diameter of the gas vesicles may range from 10 nm to 1000nm, such as 25 nm to 500 nm, including 50 nm to 250 nm, or 100 nm to 250nm. By “average” is meant the arithmetic mean.

GVs in the sense of the disclosure have different shapes depending ontheir genetic origins [7]. For example, GVs in the sense of thedisclosure can be substantially spherical, ellipsoid, cylindrical, orhave other shapes such as football shape or cylindrical with cone shapedend portions depending on the type of bacteria providing the gasvesicles.

In embodiments herein described, GVs in the sense of the disclosure arecapable of withstanding pressures of several kPa. but collapseirreversibly at a pressure at which the GV protein shell is deformed tothe point where it flattens or breaks, allowing the gas inside the GV todissolve irreversibly in surrounding media, herein also referred to as acritical collapse pressure, or selectable critical collapse pressure, asthere are various points along a collapse pressure profile.

A collapse pressure profile as used herein indicates a range ofpressures over which collapse of a population of GVs of a certain typeoccurs. In particular, a collapse pressure profile in the sense of thedisclosure comprise increasing acoustic/hydrostatic collapse pressurevalues, starting from an initial collapse pressure value at which the GVsignal/optical scattering by GVsstarts to be erased to a completecollapse pressure value at which the GV signal/optical scattering by GVsis completely erased. The collapse pressure profile of a set type of GVis thus characterized by a mid-point pressure where 50% of the GVs ofthe set type have been collapsed (also known as the “midpoint collapsepressure”), an initial collapse pressure where 5% or lower of the GVs ofthe type have been collapsed, and a complete collapse pressure where atleast 95% of the GVs of the type have been collapsed. In embodimentsherein described a selectable critical collapse pressure can be any ofthese collapse pressures within a collapse pressure profile, as well asany point between them. The critical collapse pressure profile of a GVis functional to the mechanical properties of the protein shell and thediameter of the shell structure. The profiles under hydrostatic pressureand under acoustic pressure are different, with the points on theacoustic pressure profile being higher in pressure than the hydrostaticprofile at the midpoint collapse pressure point, at least.

In embodiments herein described, it has been surprisingly found that thecritical collapse pressure is also functional to the manner in which theforces are applying the pressure to the GV shell. Accordingly, differentways of applying pressure on a set GVs result in different types ofcritical collapse pressures associated to the set GV. As a consequence,GVs in the sense of the disclosure are associated to more than onecritical collapse pressure profile, depending on whether the pressure onthe GV is applied in a hydrostatic manner (hydrostatic pressure), orapplied in an acoustic manner (acoustic pressure).

The term “hydrostatic pressure” as used herein indicates the pressureexerted by a fluid at a given point within the fluid, absent fluidmotion. Hydrostatic pressure includes pressure due to gravity, whichpressure increases in proportion to depth measured from the surfacebecause of the increasing weight of fluid exerting downward force fromabove. In addition, the hydrostatic pressure may include pressure due toforces applied to the fluid by solid surfaces adjoining the fluid, or byanother fluid, such as a gas. As used herein, hydrostatic pressure doesnot include pressure due to sound waves.

The term the “acoustic pressure” as used herein indicates the pressureexerted by a sound wave, such as ultrasound wave, propagating through amedium. In ultrasound imaging, this wave is typically generated by anultrasound transducer, and the pressure resulting at any time and pointin the medium is determined by transducer output and patterns ofconstructive and destructive interference, attenuation, reflection,refraction and diffraction. Ultrasound images are generated bytransmitting one or more pulses into the medium and acquiringbackscattered signals from the medium, which depend on mediumcomposition, including the presence of contrast agents.

Accordingly, in embodiments herein described each GV type has ahydrostatic collapse pressure profile and an acoustic collapse pressureprofile.

It has been surprisingly found that in GVs according to the presentdisclosure the acoustic collapse pressure is higher than the hydrostaticcollapse pressure. In particular, the acoustic collapse pressure profileof a given GV type is always shifted to higher pressures compared to itshydrostatic collapse pressure profile. The approximate mid-pointacoustic collapse pressure, Pa, can be related to the mid-pointhydrostatic collapse pressure, Ph, using a linear expression. Thislinear expression includes a non-zero positive constant, C, and apositive slope, M, such thatPa=C+M*Ph  (1)

In embodiments, herein described, for a given GV type, a Pa can bepredicted to within ±10% error from a measured Ph value, using theparameters C=475 and M=0.64. An even more precise prediction can be madefor GVs that share a substantially similar shell structure. For example,for GVs based on the Ana GV shell structure and modified by alteringGvpC composition, C=396 and M=0.80 results in a prediction of Pa withinan error of ±6% from Ph (see Example 27).

In embodiments, herein described, for a given GV type, the spread of theacoustic collapse pressure profile can similarly be predicted from thespread of the hydrostatic collapse pressure profile using a linearrelationship with positive constant C and positive slope M, such thatΔPa=C+M*ΔPh. In particular, for a ΔPh value measured for a given GVtype, the corresponding Pa can be predicted to within 30 kPa using theparameters C=6.32 and M=1.15.

In embodiments herein described, the hydrostatic collapse pressure of aparticular GV can be approximated by a sigmoidal function with a definedmid-point and transition width. For example, it can be defined accordingto Equation (2).ƒ(p)=(1+e ^((p-p) ^(c) ^()/Δp))⁻¹  (2)with p_(c) defined as the mid-point and Δp defined as the transitionwidth. These parameters are determined for each GV type by measuringcollapse as a function of pressure and fitting the resulting data withthis equation, or predicted based on the GV's molecular characteristics.

For example, the hydrostatic collapse pressure can be measured bydetecting a hydrostatic collapse behavior of the GV structures usingpressurized absorbance spectroscopy, in which the optical density of GVsis measured under increasing hydrostatic pressure (see FIG. 4 Panel b;Example 21)

In embodiments herein described, the collapse behavior of GVs underultrasound exhibits a spectral pattern, as the GVs can collapse over arange or spectra of continuous increasing acoustic collapse pressurevalues, starting from an initial collapse pressure value at which the GVsignal starts to be erased to a complete collapse pressure value atwhich the GV signal is completely erased.

The acoustic collapse pressures of a given GV type can be characterizedby an acoustic collapse pressure profile, which is a normalized sigmoidfunction f(p) defined as follows:ƒ(p)=(1+e ^((p-p) ^(c) ^()/Δp))⁻¹  (3)where p is the applied pressure, p_(c) is the collapse mid-point and Δpis the variance, the latter two being parameters obtained from fittingwith a sigmoid function. The acoustic collapse pressure profile showsnormalized ultrasound signal intensities as a function of increasingpressures.

The acoustic collapse pressure profile of a given GV type can bedetermined by imaging GVs with imaging ultrasound energy aftercollapsing portions of the given GV type population with a collapsingultrasound energy (e.g. ultrasound pulses) with increasing peak positivepressure amplitudes to obtain acoustic pressure data point of acousticpressure values, the data points forming an acoustic collapse curve. Theacoustic collapse pressure function f(p) can be derived from theacoustic collapse curve by fitting the data with a sigmoid function suchas a Boltzmann sigmoid function (see Example 21). Exemplary acousticcollapse pressure curves construed accordingly for a set of threedifferent GVs are shown in FIG. 4, panel c).

Accordingly, acoustic collapse pressure profile in the sense of thedisclosure include a set of initial collapse pressure values, a midpointcollapse pressure value and a set of complete collapse pressure values.The initial collapse pressures are the acoustic collapse pressures atwhich 5% or less of the GV signal is erased. A midpoint collapsepressure is the acoustic collapse pressure at which 50% of the GV signalis erased. Complete collapse pressures are the acoustic collapsepressures at which 95% or more of the GV signal is erased.

The initial collapse pressures can be obtained by solving the fittedequations for p such that ƒ(p)≤0.05. The midpoint collapse pressure canbe obtained by solving the fitted equations for p such that ƒ(p)=0.5.The complete collapse pressures can be obtained by solving the fittedequations for p such that ƒ(p)≥0.95.

In particular, for a set GV an acoustic collapse pressure can be morethan twice the hydrostatic collapse pressure for the same GV as shown bya comparison of corresponding curves built based on the detectedacoustic collapse pressure values according equations (3) and ondetected hydrostatic collapse pressure value according to Equation (2).

Accordingly, in methods and systems of the present disclosure andrelated compositions and contrast agents, identification of the acousticcollapse pressure for a set GV can be used to apply an acoustic pressurewhich allows sensitive and specific detection of the set GV inultrasound imaging.

In particular, in some embodiments herein described a method to providean ultrasound image of a target site comprises: collapsing the GVPS typeby applying collapsing ultrasound to the target site, the collapsingultrasound applied at a collapsing ultrasound pressure greater than theselectable acoustic collapse pressure value. The method furthercomprises imaging the target site by applying imaging ultrasound to thetarget site, the imaging ultrasound applied at a first imagingultrasound pressure selected to provide an uncontrasted image of thetarget site.

In some embodiments, the collapsing ultrasound pressure is higher thanthe midpoint of the hydrostatic collapse pressure profile.

The method can further comprise administering to a target site acontrast agent comprising a gas vesicle protein structure hereindescribed and imaging the target site by applying imaging ultrasound tothe target site prior to the collapsing, the ultrasound applied at asecond imaging ultrasound pressure lower than the acoustic collapsepressure value and selected to provide a visible image of the targetsite. Application of an ultrasound or acoustic pressure at the indicatedimaging value increases sensitivity of visualization of the related GVas it minimizes the acoustic collapse of the GV in the contrast agent.

Applying ultrasound refers to sending ultrasound-range acoustic energyto a target. The sound energy produced by the piezoelectric transducercan be focused by beamforming, through transducer shape, lensing, or useof control pulses. The soundwave formed is transmitted to the body, thenpartially reflected or scattered by structures within a body; largerstructures typically reflecting, and smaller structures typicallyscattering. The return sound energy reflected/scattered to thetransducer vibrates the transducer and turns the return sound energyinto electrical signals to be analyzed for imaging. The frequency andpressure of the input sound energy can be controlled and are selectedbased on the needs of the particular imaging task and, in some methodsdescribed herein, collapsing GVs. To create images, particularly 2D and3D imaging, scanning techniques can be used where the ultrasound energyis applied in lines or slices which are composited into an image.

In some embodiments, the ultrasound imaging herein described comprisingcollapsing the GVPS type in the contrast agent by applying collapsingultrasound to the target site, the collapsing ultrasound applied at acollapsing ultrasound pressure greater than the selectable acousticcollapse pressure value. As used herein, the term “selectable acousticcollapse pressure” refers to an acoustic collapse pressure value thatcan be selected from the acoustic collapse profile of the GVPS type.

In some embodiments, imaging the target site can be performed byscanning an ultrasound image of the target site in a subject. In somecases, imaging the target site includes transmitting an imagingultrasound signal from an ultrasound transmitter to the target site, andreceiving a set of ultrasound data at a receiver. The visible image isformed by ultrasound signals backscattered from the target site. Theultrasound data can be analyzed using a processor, such as a processorconfigured to analyze the ultrasound data and produce an ultrasoundimage from the ultrasound data. In certain embodiments, the ultrasounddata detected by the receiver includes an ultrasound signal, anultrasound signal reflected by the target site of the subject.

In certain embodiments, the method includes applying a set of imagingpulses from an ultrasound transmitter to the target site, and receivingultrasound signal at a receiver. In certain instances, the ultrasoundsignal detected by the receiver includes an ultrasound echo signal.Additional information of ultrasound systems and methods can be found inrelated publications as will be understood by a person skilled in theart.

Methods for performing ultrasound imaging are known in the art and canbe employed in methods of the current disclosure. In certain aspects, anultrasound transducer, which comprises piezoelectric elements, transmitsan ultrasound imaging signal (or pulse) in the direction of the targetsite. Variations in the acoustic impedance (or echogenicity) along thepath of the ultrasound imaging signal causes backscatter (or echo) ofthe imaging signal, which is received by the piezoelectric elements. Thereceived echo signal is digitized into ultrasound data and displayed asan ultrasound image. Conventional ultrasound imaging systems comprise anarray of ultrasonic transducer elements that are used to transmit anultrasound beam, or a composite of ultrasonic imaging signals that forma scan line. The ultrasound beam is focused onto a target site byadjusting the relative phase and amplitudes of the imaging signals. Theimaging signals are reflected back from the target site and received atthe transducer elements. The voltages produced at the receivingtransducer elements are summed so that the net signal is indicative ofthe ultrasound energy reflected from a single focal point in thesubject. An ultrasound image is then composed of multiple image scanlines.

In some embodiments, imaging the target site is performed by applying ortransmitting an imaging ultrasound signal from an ultrasound transmitterto the target site and receiving a set of ultrasound data at a receiver.The ultrasound data can be obtained using a standard ultrasound device,or can be obtained using an ultrasound device configured to specificallydetect the contrast agent used. Obtaining the ultrasound data caninclude detecting the ultrasound signal with an ultrasound detector. Insome embodiments, the imaging step further comprises analyzing the setof ultrasound data to produce an ultrasound image.

In certain embodiments, the ultrasound signal has a transmit frequencyof at least 1 MHz, 5 MHz, 10 MHz, 20 MHz, 30 MHz, 40 MHz or 50 MHz. Forexample, an ultrasound data is obtained by applying to the target sitean ultrasound signal at a transmit frequency from 4 to 11 MHz, or at atransmit frequency from 14 to 22 MHz. In some instances, the imagingfrequency is selected so as to maximize the contrast generated by theadministered contrast agent.

In the embodiments herein described, the collapsing ultrasound andimaging ultrasound are selected to have a collapsing pressure and animaging pressure amplitude based on the acoustic collapse pressureprofile of the GVPS type used in the contrast agent. In some instances,the ultrasound pressure, including the collapsing ultrasound pressureand the imaging ultrasound pressure can be referred to as the “peakpositive pressure” of the ultrasound pulses. The term “peak positivepressure” refers to the maximum pressure amplitude of the positive pulseof a pressure wave, typically in terms of the difference between thepeak pressure and the ambient pressure at the location in the person orspecimen that is being imaged.

In some embodiments, the collapsing ultrasound transmit pulses areselected to have a peak positive pressure amplitude equal to or higherthan an initial collapse pressure in the acoustic collapse profile ofthe GVPS used in the contrast agent administered to the target site.

In some embodiments, the collapsing ultrasound transmit pulses areselected to have a peak positive pressure amplitude equal to or higherthan the midpoint collapse pressure in the acoustic collapse profile ofthe GVPS used in the contrast agent administered to the target site.

In some embodiments, the collapsing ultrasound transmit pulses areselected to have a peak positive pressure amplitude equal to or higherthan a complete collapse pressure in the acoustic collapse profile ofthe GVPS used in the contrast agent administered to the target site.

In some embodiments, collapsing the GV type is followed by imaging thetarget site by applying imaging ultrasound to the target site, theimaging ultrasound applied at a first imaging ultrasound pressureselected to provide an uncontrasted image of the target site.

In some embodiments, the imaging ultrasound transmit pulses are selectedto have a peak positive pressure amplitude equal to or lower than aninitial collapse pressure in the acoustic collapse profile of the GVPSused in the contrast agent administered to the target site.

In some embodiments, the imaging ultrasound transmit pulses are selectedto have a peak positive pressure amplitude equal to or lower than amidpoint collapse pressure in the acoustic collapse profile of the GVPSused in the contrast agent administered to the target site.

In some embodiments, the imaging ultrasound transmit pulses are selectedto have a peak positive pressure amplitude equal to or lower than acomplete collapse pressure in the acoustic collapse profile of the GVPSused in the contrast agent administered to the target site.

In some embodiments, prior to collapsing the GV type, the ultrasoundimaging method further comprises imaging the target site by applyingimaging ultrasound to the target site, the imaging ultrasound applied ata second imaging ultrasound pressure lower than the selectable acousticcollapse pressure and selected to provide a visible image; and comparingthe visible image of the target site with the uncontrasted image. Insome embodiments, different ultrasound pressure values can be used forimaging at different steps of the process. In other embodiments, thepressure used for imaging can be constant throughout the method.

In some embodiments, the collapsing ultrasound pressure is selected tobe equal to or higher than the midpoint collapse pressure, preferablyequal to or higher than the complete collapse pressure, in the acousticcollapse profile of the GV, and the first and second imaging ultrasoundpressure is equal to or lower than the midpoint collapse pressure,preferably equal to or lower than the initial collapse pressure, in theacoustic collapse profile of the GV.

In some embodiments, the collapsing ultrasound pressure used to collapsethe GVPS type is higher than the midpoint of the hydrostatic collapsepressure profile of the GVPS type.

In some embodiments, imaging the target site using ultrasound imagingmethod comprises applying a first imaging ultrasound signal having afirst imaging peak positive pressure from an ultrasound transmitter tothe target site, receiving a first set of ultrasound data at a receiver,and analyzing the first set of ultrasound data to produce a firstultrasound image; applying a collapsing ultrasound signal having a firstcollapsing peak positive pressure from the ultrasound transmitter to thetarget site; applying a second imaging ultrasound signal having a secondimaging peak positive pressure from the ultrasound transmitter to thetarget site, receiving a second set of ultrasound data at the receiver,analyzing the second set of ultrasound data to produce a secondultrasound image; and processing the first and second set of ultrasounddata.

In particular, the first imaging peak positive pressure and the secondimaging peak positive pressure can be the same and both lower than theinitial collapse pressure of the GV used in the contrast agent, and thecollapsing peak positive pressure is higher than the complete collapsepressure of the GV. In such case, the first image represents acontrasted image of the target site containing the uncollapsed GVs andthe second image represents uncontrasted image of the target sitecontaining the collapsed GVs. The processing the first and second set ofultrasound data involves subtracting the second image from the firstimage.

In some embodiments, the method further comprises obtaining the acousticcollapse pressure profile of the GVPS type administered to the targetsite. The acoustic collapse pressure profile of the GVPS type can beobtained by imaging GVPS, in vivo or in vitro, with imaging ultrasoundenergy after collapsing portions of the given GVPS type population witha collapsing ultrasound energy (e.g. ultrasound pulses) with increasingpeak positive pressure amplitudes and constructing an acoustic pressureprofile for the GVPS type. Alternatively, the acoustic collapse pressureprofile can be obtained from the hydrostatic collapse pressure profileof the GVPS type according to equation (1).

In some embodiments, the GVPS type can be wild-type GVPS from anybacterial origin or variants thereof. For example, the GVPS type can bea wild-type Ana GV containing the wild-type GvpC having an acousticcollapse pressure profile defined by an initial collapse pressure of 650kPa and a complete collapse pressure of 1,100 kPa. The first and secondimaging ultrasound pulses can be acquired with a transmit pressure below650 kPa and the collapsing ultrasound pulses can be acquired with atransmit pressure above 1,100 kPa.

In methods herein described, administering the contrast agent can beperformed in any way suitable to deliver a GV to the target site to beimaged. In some embodiments, the contrast agent can be administered tothe target site locally or systemically.

The wording “local administration” or “topic administration” as usedherein indicates any route of administration by which a GV is brought incontact with the body of the individual, so that the resulting GVlocation in the body is topic (limited to a specific tissue, organ orother body part where the imaging is desired). Exemplary localadministration routes include injection into a particular tissue by aneedle, gavage into the gastrointestinal tract, and spreading a solutioncontaining GVs on a skin surface.

The wording “systemic administration” as used herein indicates any routeof administration by which a GV is brought in contact with the body ofthe individual, so that the resulting GV location in the body issystemic (i.e. non limited to a specific tissue, organ or other bodypart where the imaging is desired). Systemic administration includesenteral and parenteral administration. Enteral administration is asystemic route of administration where the substance is given via thedigestive tract, and includes but is not limited to oral administration,administration by gastric feeding tube, administration by duodenalfeeding tube, gastrostomy, enteral nutrition, and rectal administration.Parenteral administration is a systemic route of administration wherethe substance is given by route other than the digestive tract andincludes but is not limited to intravenous administration,intra-arterial administration, intramuscular administration,subcutaneous administration, intradermal, administration,intraperitoneal administration, and intravesical infusion.

Accordingly, in some embodiments of methods herein described,administering a contrast agent can be performed topically orsystemically by intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, rectal, vaginal, and oral routes.In particular, a contrast agent can be administered by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, vaginal, rectal and intestinal mucosa, etc.) and canoptionally be administered together with other biologically activeagents. In some embodiments of methods herein described, administering acontrast agent can be performed by injecting the contrast agent into asubject at the target site of interest, such as in a body cavity orlumen. In some embodiments, it can be performed by spreading a solutioncontaining the contrast agent on a region of the skin.

In some embodiments, the contrast agents are administered to the targetsite with the selected GVPS type at a sufficient concentration to bevisible by ultrasound in the context of the background tissue. Thesaturated concentration for gas vesicles are typically above 10 pM.

In some embodiments, a multiplexed ultrasound imaging method isdescribed. The term “multiplex” refers to the presence of two or moredistinct GVPS types, each of which exhibits an acoustic collapsepressure profile distinct from one another. The two or more distinctGVPSs can be derived from different bacteria or variants of GVPSs fromthe same or different bacteria.

In particular, in some embodiments, methods for multiplexed imaging of atarget site herein described comprise an ultrasound imaging method to beused on a target site contrasted with a contrast agent comprising atleast a first gas vesicle protein structure (GVPS) type and a secondGVPS type is described. In the method, the first GVPS type exhibits afirst acoustic collapse pressure profile and a first selectable acousticcollapse pressure value and a second GVPS type exhibits a secondacoustic collapse pressure profile and a second selectable acousticcollapse pressure value. GVPSEach acoustic collapse pressure profile isdefined as a collapse function from which a collapse amount can bedetermined and a different selectable acoustic collapse pressure valuecan be selected from their corresponding acoustic collapse pressureprofile.

The method comprises selectively collapsing the first GVPS type byapplying collapsing ultrasound to the target site, the collapsingultrasound applied at a first collapsing pressure value equal to orhigher than the first selectable acoustic collapse pressure value andlower than the second selectable acoustic collapse pressure value.

The method further comprises imaging the target site containing second,uncollapsed, GVPS type by applying imaging ultrasound to the targetsite, the imaging ultrasound applied at an imaging pressure value lowerthan the acoustic collapse pressure value of the second gas vesiclestructure type

In multiplexing methods herein described, both the collapsing pressureof the collapsing ultrasound and the imaging pressure of the imagingultrasound are selected based on the acoustic collapse pressure profilesof the GVPS types to selectively collapse one GVPS type over the otherGVPS types.

The term “selectively collapse” refers to collapsing at least a portionof one GVPS type in a greater amount that any other GVPS type in amixture containing a plurality of GVPS types. For any two given GVPStypes each exhibiting an acoustic pressure profile characterized byf(p), the collapsing pressure is selected to have a f1(p) value for thefirst GVPS type greater than a f2(p) value for the second GVPS type inorder to selectively collapse the first GVPS type.

In some embodiments, the collapsing pressure of the collapsingultrasound is equal to a maximally informative collapse pressure(“MIAP”) of two spectrally adjacent GV types.

The term “maximally informative collapse pressure” or “MIAP” as usedherein indicates an acoustic pressure chosen based on the acousticcollapse profiles of the two GV types such that the fraction of thefirst GV collapsed at this pressure is maximally different from thefraction of the second GV collapsed at this pressure.

Accordingly, a maximally informative acoustic pressure (MIAP) value fora set GVPS relative to another one or more GVPS types in a GVPS mixturecan be performed based on acoustic collapse profiles construed usingdetected acoustic collapse pressure values according to Equation (3). Inparticular, for GVPSs in a set GVPS mixture the MIAP can be expressed asa pressure bymaximizing Δf(p), i.e. f1(p)−f2(p), wherein ƒ1(p)=(1+e ^((p-p) ^(c)^()/Δp))⁻¹ and ƒ2(p)=(1+e ^((p-p) ^(c) ^()/Δp))⁻¹   (4)f1(p) and f2(p) corresponds to an acoustic collapse profile of the firstGVPS and the second GVPS.

The target site is then imagined following the collapsing by applying animaging ultrasound. The imaging pressure of the imaging ultrasound isselected to be equal to or lower than the acoustic collapse pressures ofremaining uncollapsed GVPS types in the target site. Preferably, theimaging pressure of the imaging ultrasound is selected to be equal to orlower than the initial acoustic collapse pressures of the remaininguncollapsed GVPS types in the target site. More preferably, the imagingpressure of the imaging ultrasound is selected to be equal to or lowerthan the lowest initial collapse pressures of all the GVPS types in thecontrast agent, including the collapsed GVPS type.

In some embodiments, the multiplexed ultrasound imaging method furthercomprises after imaging the target site containing the second,uncollapsed GVPS type, collapsing the second GVPS type by applying asecond collapsing ultrasound and imaging the target site containing thecollapsed GVPS types by applying a second imaging ultrasound. The secondcollapsing pressure is higher than the first collapsing pressure. Insome embodiments, the second collapsing pressure is higher than thecomplete collapse pressure of the second GVPS type.

In some embodiments, the multiplex ultrasound imaging method furthercomprises, prior to collapsing the first GVPS type, imaging the targetsite containing uncollapsed GVPS types, the imaging ultrasound appliedat an imaging pressure value lower than the selectable acoustic collapsepressure value for the first GVPS type, thus obtaining an imageindicative of uncollapsed first and second gas vesicle protein structuretypes.

For example, Ana GVs with wild-type GvpC (first GVPS type) and Ana GVswith GvpCΔN&C (second GVPS type) can be imaged in duplex by selectingthe MIAP to be 630 kPa. In this case, the first imaging pulse is appliedat a pressure below the initial collapse pressures of both GVPS typesand an image is acquired. Then, a second pulse is applied at 630 kPa.Then, a third pulse is applied at the same pressure as the first pulseto form the second image. Then, a fourth pulse is applied at a pressureabove the complete collapse pressure of the second GV type. Then, afifth pulse is applied at the same pressure as the first pulse to form athird image.

In some embodiments, for N number of GVPS types contained in thecontrast agent, N+1 number of ultrasound images are generated (see FIG.27). The collapsing and imaging steps are alternated so that eachcollapsing step is followed by an imaging step, in which the collapsingpressure of the collapsing ultrasound is higher than the imagingpressure of the imaging ultrasound.

In some embodiments, a multiplexed imaging method comprises thefollowing steps:

applying a base imaging ultrasound pressure from an ultrasoundtransmitter to the target site, receiving a base set of ultrasound dataat a receive, and analyzing the base set of ultrasound data to produce abase ultrasound image; the base ultrasound image represents apre-collapse baseline.

applying a first collapsing ultrasound pressure from an ultrasoundtransmitter to the target site, applying a first imaging ultrasoundpressure, receiving a first set of ultrasound data at a receiver, andanalyzing the first set of ultrasound data to produce a first ultrasoundimage; and

applying a second collapsing ultrasound pressure from an ultrasoundtransmitter to the target site, applying a second imaging ultrasoundpressure, receiving a second set of ultrasound data at a receiver, andanalyzing the second set of ultrasound data to produce a secondultrasound image.

In particular, in embodiments of methods herein described, the imagingultrasound pressures can be the same or different, preferably lower thanthe initial acoustic collapse pressures of the remaining uncollapsed GVtypes in the target site, or even more preferably lower than the lowestinitial collapse pressures of the GV types used in the contrast agent.The first and second collapsing ultrasound pressures are selected basedon the acoustic collapse pressure profiles of the GV types, with thesecond collapsing ultrasound pressure higher than the first collapsingultrasound pressure.

In certain embodiments, imaging the target site further can compriseapplying at least a third collapsing ultrasound pressure from anultrasound transmitter to the target site, applying a third imagingultrasound pressure, receiving a third set of ultrasound data at areceiver, and analyzing the third set of ultrasound data to produce athird ultrasound image. The third collapsing ultrasound pressure isselected to be higher than the second collapsing ultrasound pressure,preferably higher than the complete collapse pressure of the third GVPStype.

In some embodiments, the multiplexed ultrasound imaging method furthercomprises obtaining a first acoustic collapse pressure profile of afirst GV and at least a second acoustic collapse pressure profile of atleast a second GV, and calculating a first maximally informativecollapse pressure from the obtained first and second acoustic collapsepressure profiles. Each acoustic collapse pressure profile ischaracterized by a fitted sigmoid function f(p) as described above.

In some embodiments, the multiplexing ultrasound imaging method furthercomprises obtaining a third acoustic collapse pressure profile of athird GV, and calculating a second maximal informative collapse pressurefrom the obtained second and third acoustic collapse pressure profiles.

In some embodiments, the multiplexed ultrasound imaging method furthercomprises applying a base imaging ultrasound pressure having a base peakpositive pressure lower than the lowest initial collapse pressure of theGVPS types, receiving a base set of ultrasound data at a receive, andanalyzing the base set of ultrasound data to produce a base ultrasoundimage;

selectively collapsing the first GVPS type by applying collapsingultrasound having a first collapsing pressure amplitude equal to thefirst maximal informative collapse pressure, imaging the target site byapplying imaging ultrasound having a first imaging pressure, receiving afirst set of ultrasound data at a receiver, and analyzing the first setof ultrasound data to produce a first ultrasound image;selectively collapsing the second GVPS by applying collapsing ultrasoundhaving a second collapsing pressure amplitude equal to the secondmaximal informative collapse pressure, imaging the target site byapplying imaging ultrasound having a second imaging pressure, receivinga second set of ultrasound data at the receiver, and analyzing thesecond set of ultrasound data to produce a first ultrasound image;selectively collapsing the third GVPS by applying collapsing ultrasoundhaving a third collapsing pressure amplitude greater than the highestcomplete collapse pressure of the GVPSs, imaging the target site byapplying imaging ultrasound having a third imaging pressure, receiving athird set of ultrasound data at the receiver, and analyzing the thirdset of ultrasound data to produce a third ultrasound image; andprocessing the produced images.

In particular, the first, second and third imaging ultrasound pressurecan be equal to the base imaging ultrasound pressure, which are lowerthan the lowest initial collapse pressure of the GVPS types administeredto the target site.

In certain embodiments of multiplexed methods herein described, imaginga target site can be performed by ummixing the ultrasound signal of oneor more GVPS types using multiple ultrasound signals selected to provideto the target site a suitable acoustic pressure directed to selectivelyimage and/or collapse one or more GVPS types of a plurality of GVPStypes administered to the target site.

For example, in a contrast agent comprising ΔGvpC, ΔN&C and GvpC_(WT),the first collapsing pressure is equal to the first maximal informativeacoustic pressure of 630 kPa calculated based on the acoustic collapseprofiles of ΔGvpC variant and ΔN&C variant. The first maximalinformative acoustic pressure is capable of maximally collapsing theΔGvpC variant while minimally collapsing the other two variants, i.e.ΔN&C and GvpC_(WT). The second collapsing pressure is equal to thesecond maximal informative acoustic pressure of 790 kPa calculated basedon the acoustic collapse profiles of ΔN&C variant and GvpC_(WT). Thesecond maximal informative acoustic pressure is capable of maximallycollapsing the ΔN&C variant while minimally collapsing the remainingvariant, i.e. GvpC_(WT). The third collapsing pressure is about 1230kPa, higher than the complete collapse pressure of GvpC_(WT) in order tocollapse the remaining GvpC_(WT) variant.

In some embodiments, the multiplexing ultrasound imaging method furthercomprises obtaining an acoustic pressure profile of each GVPS typeadministered to the target site. The acoustic collapse pressure profileof each GVPS type can be obtained by imaging the GVPS, in vivo or invitro, with imaging ultrasound energy after collapsing portions of thegiven GVPS type population with a collapsing ultrasound energy (e.g.ultrasound pulses) with increasing peak positive pressure amplitudes andconstructing an acoustic collapse pressure profile for the GVPS type.Alternatively, the acoustic collapse pressure profile can be obtainedfrom a hydrostatic collapse pressure profile of the GVPS type accordingto equation (1).

In some embodiments, a multiplexing ultrasound imaging a target sitecontrasted with a plurality of gas vesicle protein structure (GVPS)types can be performed with a multiplex method using a plurality of GVstypes. In the method, each GVPS type exhibits i) an acoustic collapsepressure profile defined as a collapse function from which a collapseamount can be determined, and ii) a selectable acoustic collapsepressure value, selectable acoustic collapse pressure values going froma lowest acoustic collapse pressure value to a highest acoustic collapsepressure value. the method comprises selectively collapsing GVPS type toa collapse amount higher than a collapse amount of each remaining GVPStype by applying collapsing ultrasound to the target site, thecollapsing ultrasound applied at a pressure value equal to or higherthan the selectable acoustic collapse pressure value of the GVPS typebeing collapsed and lower than an acoustic collapse pressure value ofsaid each remaining GVPS type or types. The method further comprisesimaging the target site containing the remaining GVPS type or types byapplying imaging ultrasound to the target site, the imaging ultrasoundapplied at a pressure value lower than a lowest acoustic collapsepressure value of said each remaining GVPS type or types. The methodalso comprises repeating the collapsing and the imaging until all GVPStypes are collapsed, thus providing a sequence of visible images of thetarget site, the sequence being indicative of image-by-image decreasingremaining GVPS types.

In the embodiments herein described, the multiplexed ultrasound imagingmethod further comprises an initial preparation step of administering tothe target site a contrast agent comprising the plurality of gas vesicleprotein structure types.

In those embodiments, imaging the target site further comprisesprocessing the produced images using acoustic spectral unmixing toobtain spectrally unmixed images (Example 21). The term “acousticspectral unmixing” or “pressure spectral unmixing” refers to amathematical image processing method for obtaining spectrally unmixedimages by subtracting each sub-population of signals from a sum ofsignal contributed by each sub-population present in any given pixel.

In those embodiments that the total signal for a mixed population of GVsin any given pixel is the sum of signals contributed by eachsub-population present in that pixel. By acquiring images whilesequentially applying collapse pulses of increasing pressure (P_(i)),the change in the pixel-wise signal intensity (I) between differentpulses contains information about the abundance of each GV type in thepixel (FIG. 4, panel e). This information is extracted by multiplyingthe measured differential signalsΔ_(i) =I(P _(i-1))−I(P _(i))  (5)by the inverse of a matrix containing the differential collapse profileof each type of GV, denoted by α_(i,j). For GV type j, the applicationof pressure P, in a specific sequence of applied pressures results inthe collapse of a fraction of that GV type corresponding to α_(i,j). Thevalue of α_(i,j) for a given GV type is calculated from thecorresponding acoustic collapse profile described by a sigmoidalfunction f(p)_(j), such that α_(I,j) is the difference between 1 and thefraction of GVs intact at P₁, and α_(2,j) is the difference between thefraction of GVs intact at P₁ and the fraction of GVs intact at P₂, andso on. More generally, α_(i,j) is the difference between the fraction ofGVs intact at P_(i-1) and the fraction of GVs intact at P_(i).

The contribution of each GV type to the observed signal, represented asC₁, is given by the matrix operation:C=α ⁻¹Δ.  (6)

In some embodiments, the GVs can be engineered to modulate the GVmechanical, acoustic, surface and targeting properties in order toachieve enhanced harmonic responses and multiplexed imaging to be betterdistinguished from background tissues.

In embodiments herein described Gas vesicles protein structures can beprovided by Gyp genes endogenously expressed in bacteria or archea.Endogenous expression refers to expression of Gyp proteins forming theprotein shell of the GV in bacteria or archaea that naturally producegas vesicles encoded (e.g. in their genome or native plasmid DNA).

Gyp proteins expressed by bacteria or archea typically include twoprimary structural proteins, here also indicated as GvpA and GvpC, andseveral putative minor components and chaperones [2, 8, 9] as would beunderstood by a person skilled in the art.

Reference is made to the illustration of FIG. 1 showing a schematicrepresentation of the structure of a GV. In the illustration of FIG. 1GvpA and GvpC are indicated as the two major structural constituents ofGVs, with GvpA ribs (1) (gray) forming the primary GV shell and theouter scaffold protein GvpC (2) (black) conferring structural integrity.In particular, in the illustration of FIG. 1, the light gray elementsrepresent the proteinaceous gas vesicle shell, comprising multiplecopies of GvpA and other minor structural constituents. In theillustration of FIG. 1, the dark rectangles (2) bound to the surface ofthe gas vesicle shell represent GvpC, a protein that affects mechanicaland acoustic properties of the gas vesicle.

In bacteria or archaea expressing GVs, the Gyp proteins forming a GV'sprotein shell are encoded by a cluster of 8 to 14 different genesdepending on the host bacteria or archaea, as will be understood by askilled person.

The term “gene cluster” as used herein means a group of two or moregenes found within an organism's DNA that encode for two or morepolypeptides or proteins, which collectively share a generalizedfunction or are genetically regulated together to produce a cellularstructure and are often located within a few thousand base pairs of eachother. The size of gene clusters can vary significantly, from a fewgenes to several hundred genes [10]. Portions of the DNA sequence ofeach gene within a gene cluster are sometimes found to be similar oridentical; however, the resulting protein of each gene is distinctivefrom the resulting protein of another gene within the cluster. Genesfound in a gene cluster can be observed near one another on the samechromosome or native plasmid DNA, or on different, but homologouschromosomes. An example of a gene cluster is the Hox gene, which is madeup of eight genes and is part of the Homeobox gene family. In the senseof the disclosure, gene clusters as described herein also comprise gasvesicle gene clusters, wherein the expressed proteins thereof togetherare able to form gas vesicles.

In embodiments herein described, identification of a gene clusterencoding for Gyp proteins in a bacteria or archaea can be performed forexample by isolating the GVs from the bacteria or archaea, isolating theprotein for the protein shell of the GV and derive the related aminoacidic sequence with methods and techniques identifiable by a skilledperson. The sequence of the genes encoding for the Gyp proteins can thenbe identified by method and techniques identifiable by a skilled person.For example, gas vesicle gene clusters can also be identified by personsskilled in the art by performing gene sequencing or partial- orwhole-genome sequencing of organisms using wet lab and in silicomolecular biology techniques known to those skilled in the art. Asunderstood by those skilled in the art, gas vesicle gene clusters can belocated on the chromosomal DNA or native plasmid DNA of microorganisms.After performing DNA or cDNA isolation from a microorganism, thepolynucleotide sequences or fragments thereof or PCR-amplified fragmentsthereof can be sequenced using DNA sequencing methods such as Sangersequencing, DNASeq, RNASeq, whole genome sequencing, and other methodsknown in the art using commercially available DNA sequencing reagentsand equipment, and then the DNA sequences analyzed using computerprograms for DNA sequence analysis known to skilled persons.

Gas vesicle gene cluster genes [2, 8, 9] can also be identified in DNAsequence databases such as GenBank, EMBL, DNA Data Bank of Japan, andothers. Gas vesicle gene cluster gene sequences in databases such asthose above can be searched using tools such as NCBI Nucleotide BLASTand the like, for gas vesicle gene sequences and homologs thereof, usinggene sequence query methods known to those skilled in the art.

Exemplary genes present in the gene cluster for haloarchael GVs (whichhave the largest number of different gyp genes) and their predictedfunction and features are illustrated in Example 26.

Representative examples of endogenously expressed GVs are the gasvesicle protein structure produced by the Cyanobacterium Anabaenafloc-aquae (Ana GVs) [3], and the Halobacterium Halobacterium salinarum(Halo GVs) [2]. In particular, Ana GVs are cone-tipped cylindricalstructures with a diameter of approximately 140 nm and length of up to 2and in particular 200-800 nm or longer, encoded by a cluster of ninedifferent genes, including the two primary structural proteins, GvpA andGvpC, and several putative minor components and putative chaperones[11]as would be understood by a person skilled in the art. Halo GVs aretypically spindle-like structures with a maximal diameter ofapproximately 250 nm and length of 250-600 nm, encoded by a cluster offourteen different genes, including the two primary structural proteins,GvpA and GvpC, and several putative minor components and putativechaperones [11] as would be understood by a person skilled in the art

In embodiments herein described Gas vesicles protein structures can beprovided by Gyp genes heterologously expressed in bacteria or archaea.Heterologous expression refers to expression of Gyp proteins in anyspecies that either does not naturally produce gas vesicles, or whereits natural production of gas vesicles has been suppressed, for examplethrough genetic knock-out of the genes encoding Gyp proteins, and whereforeign DNA encoding gas vesicle genes is introduced into the organismto persist as a plasmid or integrate into the genome.

In some embodiments, heterologously expressed Gyp genes can comprisegenes encoding for corresponding Gyp proteins which are naturallyoccurring or have sequences having at least 50% identity with naturallyoccurring Gyp proteins.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to the nucleotidebases or residues in the two sequences that are the same when alignedfor maximum correspondence over a specified comparison window. Whenpercentage of sequence identity or similarity is used in reference toproteins, it is recognized that residue positions which are notidentical often differ by conservative amino acid substitutions, whereamino acid residues are substituted with a functionally equivalentresidue of the amino acid residues with similar physiochemicalproperties and therefore do not change the functional properties of themolecule.

A functionally equivalent residue of an amino acid used herein typicallyrefers to other amino acid residues having physiochemical andstereochemical characteristics substantially similar to the originalamino acid. The physiochemical properties include water solubility(hydrophobicity or hydrophilicity), dielectric and electrochemicalproperties, physiological pH, partial charge of side chains (positive,negative or neutral) and other properties identifiable to a personskilled in the art. The stereochemical characteristics include spatialand conformational arrangement of the amino acids and their chirality.For example, glutamic acid is considered to be a functionally equivalentresidue to aspartic acid in the sense of the current disclosure.Tyrosine and tryptophan are considered as functionally equivalentresidues to phenylalanine. Arginine and lysine are considered asfunctionally equivalent residues to histidine.

A person skilled in the art would understand that the similarity betweensequences is typically measured by a process that comprises the steps ofaligning the two polypeptide or polynucleotide sequences to form alignedsequences, then detecting the number of matched characters, i.e.characters similar or identical between the two aligned sequences, andcalculating the total number of matched characters divided by the totalnumber of aligned characters in each polypeptide or polynucleotidesequence, including gaps. The similarity result is expressed as apercentage of identity.

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparison,and multiplying the result by 100 to yield the percentage of sequenceidentity.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length protein or protein fragment. A reference sequence cancomprise, for example, a sequence identifiable a database such asGenBank and UniProt and others identifiable to those skilled in the art.

As understood by those skilled in the art, determination of percentidentity between any two sequences can be accomplished using amathematical algorithm. Non-limiting examples of such mathematicalalgorithms are the algorithm of Myers and Miller [12], the localhomology algorithm of Smith et al. [13]; the homology alignmentalgorithm of Needleman and Wunsch [14]; the search-for-similarity-methodof Pearson and Lipman [15]; the algorithm of Karlin and Altschul [16],modified as in Karlin and Altschul [17]. Computer implementations ofthese mathematical algorithms can be utilized for comparison ofsequences to determine sequence identity. Such implementations include,but are not limited to: CLUSTAL in the PC/Gene program (available fromIntelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0)and GAP, BESTFIT, BLAST, FASTA [15], and TFASTA in the WisconsinGenetics Software Package, Version 8 (available from Genetics ComputerGroup (GCG), 575 Science Drive, Madison, Wis., USA). Alignments usingthese programs can be performed using the default parameters.

In some embodiments, heterologously expressed Gyp proteins to provide aGV type have independently at least 50% sequence identity, preferably atleast 80%, more preferably at least 90%, most preferably at least 95%sequence identity compared to a reference sequence of corresponding Gypprotein using one of the alignment programs described using standardparameters.

In some exemplary embodiments, the wild-type or native GVs used hereincan be produced by Anabaena floc-aquae (Ana GVs) with their primary GvpCprotein encoded by SEQ ID NO:2. Alternatively, the native GVs usedherein can be derivatives of native GVs having one or more variationsincluding insertions, deletions or replacement with at least 30%preferably at least 80% identity with respect to SEQ ID NO:2 and anE-value of less than 0.00001. GvpC proteins from other microorganismscan also be used as a reference sequence including the sequences listedin Table 4.

Heterologous expression of GVs in bacteria or archaea that do notexpress GVs can be performed by cloning one or more polynucleotidesencoding naturally occurring Gyp proteins or homologs thereof that arerequired for production of GVs (comprising gvpA, gvpC, and otherproteins known to those skilled in the art and described herein) intoone or more suitable expression plasmids or vectors, and expressing theheterologous GV proteins in the bacteria or archaea. Polynucleotidesencoding GV protein genes can be cloned using commercially availablereagents from vendors such as Qiagen, Invitrogen, Applied Biosystems,Promega, and others, following standard molecular biology methods knownin the art, such as those described herein. As would be understood bythose skilled in the art, polynucleotides encoding GV protein genes canbe obtained from several different sources. For example, polynucleotidesencoding GV proteins can be obtained by isolating genomic DNA or cDNAencoding GV proteins from microorganisms whose genomes encode GVproteins genes, and/or express GV proteins RNA. RNA can be isolated froma cell that expresses GV proteins genes, and cDNA produced by reversetranscription using standard techniques and commercial kits. Genomic DNAcan be purified from the cell, and cDNA or genomic DNA encoding one ormore GV proteins isolated, following methods known to those in the art.Alternatively, polynucleotides comprising one or more gas vesicle genescan be synthesized using oligonucleotide and polynucleotide syntheticmethods known in the art. PCR-based amplification of one or more GVprotein genes can be performed using appropriately designed primer pairs(e.g. using PrimerDesign or other programs known to those skilled in theart). PCR-based amplification can be followed by ligation (e.g. using T4DNA ligase) of a polynucleotide encoding gas vesicle gene amplicon intoan appropriate expression cassette in a plasmid suitable for propagationin bacteria or other cells, such as transformation-competent E. coliDH5alpha, followed by growth of transformed cell cultures, purificationof the plasmid for confirmation of the cloned enzyme by DNA sequenceanalysis, among other methods known to those skilled in the art.Expression vectors can comprise plasmid DNA, viral vectors, or non-viralvectors, among others known to those skilled in the art, comprisingappropriate regulatory elements such as promoters, enhancers, andpost-transcriptional and post-translational regulatory sequences thatare compatible with the bacteria or archaea heterologously expressingthe GV, as would be understood by a skilled person. Promoters can beconstitutively active or inducible. Exemplary inducible expressionsystems comprise IPTG-inducible expression as described in the Examples.

In some embodiments, where one or more Gyp proteins are expressedheterologously to form GVs in microorganisms other that the native host,the related sequence can be optimized for expression in the heterologoushost microorganism as will be understood by a skilled person.

In particular, in some embodiments described herein, wherein GV isproduced heterologously production of a GV gvpc gene sequences can becodon-optimized for expression in one or more microorganism of choicesuch as Escherichia coli, according to methods identifiable by a skilledperson. As would be understood by those skilled in the art, the term“codon optimization” as used herein refers to the introduction ofsynonymous mutations into codons of a protein-coding gene in order toimprove protein expression in expression systems of a particularorganism, such as E. coli in accordance with the codon usage bias ofthat organism. The term “codon usage bias” refers to differences in thefrequency of occurrence of synonymous codons in coding DNA. The geneticcodes of different organisms are often biased towards using one of theseveral codons that encode a same amino acid over others—thus using theone codon with, a greater frequency than expected by chance. Optimizedcodons in microorganisms, such as Escherichia coli or Saccharomycescerevisiae, reflect the composition of their respective genomic tRNApool. The use of optimized codons can help to achieve faster translationrates and high accuracy.

In the field of bioinformatics and computational biology, manystatistical methods have been proposed and used to analyze codon usagebias. Methods such as the ‘frequency of optimal codons’ (Fop), theRelative Codon Adaptation (RCA) or the ‘Codon Adaptation Index’ (CAI)are used to predict gene expression levels, while methods such as the‘effective number of codons’ (Nc) and Shannon entropy from informationtheory are used to measure codon usage evenness. Multivariatestatistical methods, such as correspondence analysis and principalcomponent analysis, are widely used to analyze variations in codon usageamong genes. There are many computer programs to implement thestatistical analyses enumerated above, including CodonW, GCUA, INCA, andothers identifiable by those skilled in the art. Several softwarepackages are available online for codon optimization of gene sequences,including those offered by companies such as GenScript, EnCorBiotechnology, Integrated DNA Technologies, ThermoFisher Scientific,among others known those skilled in the art. Those packages can be usedin providing Gyp proteins with codon ensuring optimized expression invarious cell systems as will be understood by a skilled person.

A representative example of heterologous GVs is the E. coli expressing aheterologous GV gene cluster from Bacillus megaterium (Mega). Mega GVsare typically cone-tipped cylindrical structures with a diameter ofapproximately 73 nm and length of 100-600 nm, encoded by a cluster ofeleven or fourteen different genes, including the primary structuralprotein, GvpA, and several putative minor components and putativechaperones [11, 18] as would be understood by a person skilled in theart.

In some embodiments, the GVs can be engineered to modulate theirmechanical, acoustic, surface and targeting properties in order toachieve enhanced harmonic responses and multiplexed imaging to be betterdistinguished from background tissues. In particular in thoseembodiments, a GV can be engineered to provide a variant GvpC proteinand corresponding variant GV type and/or to provide a variant GV typewith a modified amount of native or engineered GvpC protein on theprotein shell of the GV.

A GvpC protein is a hydrophilic protein encoded by a gene of the GV genecluster, which includes repetitions of one repeat region flanked by anN-terminal region and a C terminal region. The term “repeat region” or“repeat” as used herein with reference to a protein refers to theminimum sequence that is present within the protein in multiplerepetitions along the protein sequence without any gaps. Accordingly, ina GvpC multiple repetitions of a same repeat is flanked by an N-terminalregion and a C-terminal region. In a same GvpC, repetitions of a samerepeat in the GvpC protein can have different lengths and differentsequence identity one with respect to another.

Repeat regions within any given GvpC sequence ‘X’ from organism ‘Y’ canbe identified by comparing the related sequence with the sequence of aknown GvpC (herein e.g. reference GvpC sequence “Z”). In particular thecomparing can be performed onby aligning sequence ‘X’ to the referenceGvpC sequence ‘Z’ using a sequence alignment tools such as BLASTP orother sequence alignment tools identifiable by a skilled person at thedate of filing of the application upon reading of the presentdisclosure. In particular, the reference sequence ‘Z’ is chosen from ahost that is the closest phylogenetic relative of ‘Y’, from a list ofAnabaena floc-aquae, Halobacterium salinarum, Haloferax mediditerranei,Microchaetae diplosiphon and Nostoc sp. The sequence alignment of ‘X’and ‘Z’ (e.g. a BLASTP) is performed by performing a first alignment ofsequence X and sequence Z to identify a beginning and an end of a repeatin ‘X as well as a number of repetition of the identified repeat, inaccordance with the known repeats in ‘Z’. The first alignment results inat least one first aligned portion of X with respect to referencesequence Z. The aligning can also comprises performing a secondalignment between the at least one first aligned portion of X identifiedfollowing the first alignment and additional portions of X to identifyat least one repeat ‘R1’ in X. Other repeats in ‘X’ (i.e. R2, R3, R4 . .. ) can subsequently be identified with respect to R1.

In performing alignment steps sequence are identified as repeat when thesequence show at least 3 or more of the following characteristics:

1) There are no gaps or spacer amino acids between any two adjacentrepetition of a repeat (see e.g. FIG. 16 and FIG. 26)

2) Each repetition of a repeat has a sequence length between 18-45 aminoacids, e.g. 33 amino acids seen for 100% of the repeats in Anabaenafloc-aquae, Microchaetae diplosiphon and Nostoc sp. (e.g. FIG. 26)

3) Upon alignment of all the repeats within a given GvpC sequence, thereexists for every position in more than 50% of the total number ofrepeats, greater than 50% sequence similarity of the amino acid residuesin each repeat (e.g. FIG. 26)

4) Sub-sequences of at least 3 or more amino acids at the beginning orend of the that are conserved across 50% or more of the repeats in agiven GvpC sequence, also referred to as “consensus sequences”.Exemplary embodiments of such consensus sequences are QAQELLAF (SEQ IDNO:32) at the end of repeats in Anabaena floc-aquae, LHQF (SEQ ID NO:33)at the end of repeats in Microchaete diplosiphon, LSQF (SEQ ID NO:34) atthe end of repeats in Microcystis aeruginosa and DAF at the beginning ofrepeats in Halobacterium salinarum. (e.g. FIGS. 16 and 26).

5) The consensus sequence of all the repeats within a given GvpCsequence show greater than 60% percent identity to the consensussequence of all the repeats within another GvpC from a differentmicrobial host of the same phylogenetic order (e.g. FIG. 26, panelsg-h).

In some exemplary embodiments, the repeat has at least 90% sequenceidentity with another repeat within the same GvpC sequence.

In a GvpC the N-terminal region comprises the amino acid residuesupstream (towards the N-terminus) of the first repeated sequence of theGvpC's repeat, while the C-terminal region comprises the amino acidresidues downstream (towards the C-terminus) of the last repeatedsequence of the GvpC's repeat.

GvpC protein is typically rich in glutamine, alanine and glutamic acidresidues, which account for >40% of the residues. In the exemplaryAnabaena flos-aqaue, GvpC comprises five highly conserved 33-amino acidrepeats with predicted alpha-helical structure, and is believed to bindacross GvpA ribs to provide structural reinforcement [3], which alignswith experimental data. In biochemical studies, removal of GvpC andtruncations to its sequence were shown to result in a reduced thresholdfor Ana GV collapse under hydrostatic pressure. In addition, previousstudies in other species have demonstrated that GvpC can toleratefusions of bacterial and viral polypeptides.

GvpC sequences in different bacteria or archaea producing GVs typicallyhave a greater than 15% sequence identity and are produced by genesfound in the gas vesicle gene cluster.

In some embodiments, a GV can be engineered to tune the related acousticproperties. In particular the engineering can be performed bygenetically engineering a GV having an acoustic collapse pressure aP₀performed to obtain a variant GV with a critical collapse pressure aP₁lower than the aP₀.

In particular, in some embodiments, a method to tune acoustic propertiesof a gas vesicle protein structure having a critical collapse pressureaP₀ comprises engineering the GV by replacing a GvpC protein of the GV'sprotein shell with: subsaturated concentrations of the GvpC proteinand/or saturated or subsaturated concentrations of a geneticallymodified GvpC protein.

In some of those embodiments, the GvpC proteins of the native GVs areremoved with methods and techniques identifiable to a skilled person.For example, the native GvpC proteins can be removed by treating the GVswith urea, as shown in Example 1 and FIG. 2, panel d, which results inGVs with their outer GvpC layer stripped but leaves the GvpA-based shellintact.

In some embodiment of methods to tune the acoustic properties of a GV, aGvpC protein of a set type GV can be replaced by wild-GvpC orgenetically modified GvpC variants in certain concentrations eithersub-saturating or saturated, such that complete surface coverage of thegvpA shell and gas vesicle strengthening does not occur.

In particular, in some embodiments, adding molar excess of gvpC:gvpAthan that found in native GVs prior to dialysis to produce GVs canincrease aP₁ up to a threshold, above which the critical collapsepressure plateaus and does not increase any further (see FIG. 5, FIG. 6and FIG. 17). Such concentration is considered as a saturatedconcentration. Conversely, in some embodiments, adding a lower molarratio of gvpC:gvpA prior to dialysis to produce GVs can decrease aP₁.

In some embodiment of methods to tune the acoustic properties of a GV,the genetically modified GvpC protein of methods and systems can bemodified by at least one of

-   -   a) a deletion of the N-terminal region, C-terminal region or        both    -   b) a deletion of 3 or more repeats, in particular starting from        the repeat adjacent to the C-terminus and moving towards the        N-terminus    -   c) a deletion of at least one repeat immediately after the        N-terminus, and    -   d) addition of amino acids such as functional tags    -   e) substitution of a sub-sequence comprising at least nine amino        acids within the GvpC sequence, wherein the substitution refers        to replacement of amino acids in the original GvpC sequence with        any other amino acid sequence, particularly with other amino        acid sequence having sequence similarity lower than 50% with        respect to the sub-sequence within the GvpC sequence,        to obtain a gas vesicle variant with a critical collapse        pressure aP1 lower than the aP₀.

In some embodiments, a deletion can comprise a deletion of up to all ofthe amino acids of an N-terminal region, one or more repeat regions, ora C-terminal region. In some embodiments, a deletion can comprise adeletion of part of one or more of an N-terminal region, a C-terminalregion, or a repeat region. For example, a deletion can comprise part ofregion 2 and part of repeat region 3, as shown in the Examples(exemplary variant N-rep2to3-C). In some embodiments, a deletion cancomprise a deletion of more than one repeat region.

In some embodiments, a deletion of a gvpC N-terminal region or aC-terminal region can produce a gvpC variant comprised in a GV having alower aP₁ than a deletion of a gvpC repeat region.

In some embodiments, a deletion of a gvpC N-terminal deletion canproduce a gvpC variant comprised in a GV having a lower aP₁ than adeletion of a gvpC C-terminal deletion.

In some embodiments, a deletion of both a gvpC N-terminal region and agvpC C-terminal region can produce a gvpC variant comprised in a GVhaving a lower aP₁ than a deletion of a gvpC N-terminal region or aC-terminal region performed individually.

In some embodiments, a deletion of one or more repeats regions that arein a position further towards the gvpC N-terminus can produce a gvpCvariant comprised in a GV having a lower aP₁ than a deletion of one ormore repeats regions that are in a position further towards the gvpCC-terminus.

In some embodiments herein described, GV variants without GvpC proteinsor with truncated or mutated GvpC proteins exhibit lower collapsepressure compared to the native GVs under both hydrostatic pressure andultrasound (Example 2).

For example, the native Ana GVs have a hydrostatic collapse pressureabout 569.85 kPa, while the Ana GV variants free of GvpC proteins andthe Ana GV variants with truncated GvpC proteins have a hydrostaticcollapse pressure about 195.30 kPa and 374.30 kPa, respectively (seeTable 5). The native Ana GVs have an acoustic collapse pressure about868.81 kPa, while the Ana GV variants free of GvpC proteins and the AnaGV variants with truncated GvpC proteins have a hydrostatic collapsepressure about 571.00 kPa and 657.04 kPa, respectively (see Table 7).

In some embodiments, GV variants without GvpC proteins or with truncatedor mutated GvpC proteins show harmonic signals several-fold higher thanthe native GVs both in vitro and in vivo.

As used herein, the term “harmonic signal” or “harmonic frequency”refers to a frequency in a periodic waveform that is an integer multipleof the frequency of the fundamental signal. In addition, this termencompasses sub-harmonic signals, which are signals with a frequencyequal to an integral submultiple of the frequency of the fundamentalsignal. In ultrasound imaging, the transmitted pulse is typicallycentered around a fundamental frequency, and received signals may beprocessed to isolate signals centered around the fundamental frequencyor one or more harmonic frequencies. In relation to the imaging of GVs,for those natural or modified GVs that are capable of producing harmonicscattering at a particular acoustic pressure, isolating receivedharmonic signals during imaging can improve the fraction of the imagesignal that is due to the GVs rather than background scattering andreflection. Exemplary GV variants showing show harmonic signals severalfold higher than the native GVs comprise GV variants such as ΔGvpC,ΔN&C-term, ΔN-term, ΔC-term, SR1, SR3, ST-GvpC, GvpC-R8, GvpC-RGD,GvpC-LRP, GvpC-mCD7, SR10ERY1, SR3CERY1, ΔN&C-CERY1, WTCERY1, GvpC-ACPP,GvpC-hPRM, N-term-rep1to2-C-term, Nterm-rep1to3-C-term,N-term-rep2to3-C-term and N-term-rep1to4-C-term. FIG. 10 shows exemplarygenetic engineering of GV surface properties, cellular targeting andmultimodal imaging. As shown in FIG. 10, gvpC genetic fusions can beused to engineer novel GV properties and functions. FIG. 11 shows anexemplary Clustal Omega sequence alignment of exemplary geneticallyengineered GvpC proteins described herein.

The term “fundamental signal” or “fundamental wave” refers to theprimary frequency of the transmitted ultrasound pulse. All GVs canbackscatter ultrasound at the fundamental frequency, allowing theirdetection by ultrasound.

The term “non-linear signal” refers to a signal that does not obeysuperposition and scaling properties, with regards to the input. Theterm “linear signal” refers to a signal that does obey those properties.One example of non-linearity is the production of harmonic signals inresponse to ultrasound excitation at a certain fundamental frequency.Another example is a non-linear response to acoustic pressure. Oneembodiment of such a non-linearity is the acoustic collapse profile ofGVs, in which there is a non-linear relationship between the appliedpressure and the disappearance of subsequent ultrasound contrast fromthe GVs as they collapse. Another example of a non-linear signal thatdoes not involve the destruction of GVs, is the increase in bothfundamental and harmonic signals with increasing pressure of thetransmitted imaging pulse, wherein certain GVs exhibit a super-linearrelationship between these signals and the pulse pressure. [19]

In some embodiments, the engineered GvpC variants are obtained byfurther linking the native GvpC protein to one or more other proteins,polypeptides, or domains to form a recombinant fusion protein.

Recombinant fusion proteins can be created artificially usingrecombinant DNA technology identifiable by a person skilled in the artof molecular biology. In general, the methods for producing recombinantfusion proteins comprise removing the stop codon from a cDNA or genomicsequence coding for the native GvpC protein having a SEQ ID NO: 2 or aderivative thereof, then appending the cDNA or genomic sequence of thesecond protein in frame through ligation or overlap extension PCR.Optionally, PCR primers can further encode a linker of one or more aminoacids residues and/or a PCR primer-encoded protease cleavage site placedbetween two proteins, polypeptides, or domains or parts thereof. Theresulting DNA sequence will then be expressed by a cell or other proteinexpression system as a single protein. A fusion protein can alsocomprise a linker of one or more amino acids residues, which can enablethe proteins to fold independently and retain functions of the originalseparate proteins or polypeptides or domains or parts thereof. Linkersin protein or peptide fusions can be engineered with protease cleavagesites that can enable the separation of one or more proteins,polypeptides, domains or parts thereof from the rest of the fusionprotein. Other methods for genetically engineering these recombinantfusion proteins include Site Directed Mutagenesis (e.g. using Q5Site-Directed Mutagenesis Kit from NEB or the QuickChange Lightning Kitfrom Agilent), Gibson Assembly (e.g. using the NEB Hi-Fi DNA AssemblyKit), Error-prone PCR (e.g. Mutazyme from Agilent) and Golden-Gateassembly (e.g. using the NEB Golden Gate Assembly Mix).

In some embodiments, the gvpC proteins described herein can besynthesized using cell-based methods or cell-free methods known to thoseskilled in the art. Protein biosynthesis can be performed by translationof DNA polynucleotides encoding the protein. Thus, protein biosynthesiscan be performed by providing cell-based or cell-free proteintranslation systems with DNA polynucleotides encoding the protein.Plasmids with genetically engineered gvpC constructs described hereincan be transformed into competent cells, such as BL21(DE3) cells(Invitrogen, Carlsbad, Calif.) or Rosetta™ (DE3)pLysS cells (MilliporeSigma, Temecula, Calif.) using electroporation, heat shock, and othermethods known to those skilled in the art and expressed in culture. ThegvpC proteins described herein can also be produced by liquid-phase orsolid-phase chemical protein synthetic methods known to those skilled inthe art [16].

In some embodiments, a gvpC variant can be produced by engineering agvpC protein from any species that encodes a gvpC protein in its genome,or a synthetically designed gvpC protein. In some embodiments, a gvpCprotein is a gvpC protein from Anabaena floc-aquae, Halobacteriumsalinarum, Halobacterium mediterranei, Microchaete diplosiphon or Nostocsp., or homologs thereof, and others identifiable by a skilled person.

In some embodiments of methods and systems and related compositionsherein described one or more GVs (including variants GVs) can beengineered to include tags peptides and/or functional group to providethe GV with additional functionalities. In particular in someembodiments GVs can be functionalized through genetic and/or chemicalmodification of a Gyp protein (including variants GvpC protein hereindescribed).

In particular, some embodiments here described tags and/or functionalgroups can be added through chemical or genetic modification of a GvpCprotein or a variant thereof in accordance with the present disclosureof a set type of GV and/or through chemical modification of another Gypprotein of the set type GV.

Reference is made to FIG. 1 showing exemplary GvpC proteins engineeredto include tags and/or functional groups. In particular, in theillustration of FIG. 1, the helical structure (3) connected to one ofthe GvpC proteins represents a genetically or chemically fused proteinfunctionality that is not present in wild-type gas vesicles. Thespherical bulb (4) connected to another GvpC represents a genetically orchemically fused fluorescent molecule allowing the gas vesicle to beimaged with both ultrasound and an optical imaging modality such asfluorescence imaging.

In some embodiments, functionalization of a Gyp protein can be performedby reacting one or more GVs with one or more compounds to allowattachment and presentation of a functional group on the protein shellof the one or more GVs.

The term “functional group” as used herein indicates specific groups ofatoms within a molecular structure that are responsible for thecharacteristic chemical reactions of that structure. Exemplaryfunctional groups include hydrocarbons, groups containing halogen,groups containing oxygen, groups containing nitrogen and groupscontaining phosphorus and sulfur all identifiable by a skilled person.In particular, functional groups in the sense of the present disclosureinclude a carboxylic acid, amine, triarylphosphine, azide, acetylene,sulfonyl azide, thio acid and aldehyde. In particular, for example, thefirst functional group and the second functional group can be selectedto comprise the following binding partners: carboxylic acid group andamine group, azide and acetylene groups, azide and triarylphosphinegroup, sulfonyl azide and thio acid, and aldehyde and primary amine.Additional functional groups can be identified by a skilled person uponreading of the present disclosure. As used herein, the term“corresponding functional group” refers to a functional group that canreact to another functional group. Thus, functional groups that canreact with each other can be referred to as corresponding functionalgroups.

The term “present” as used herein with reference to a compound orfunctional group indicates attachment performed to maintain the chemicalreactivity of the compound or functional group as attached. Accordingly,a functional group presented on a GV shell and/or a Gyp protein thereof,is able to perform under the appropriate conditions the one or morechemical reactions that chemically characterize the functional group.

In some embodiments, the functionalization of the GvpC can be performedby chemical conjugation to a GvpC protein shell of moieties such aslysine residues and/or amine-reactive crosslinkers such assulfo-N-hydroxysuccinimide esters (Sulfo-NHS). Depending on theapplication, the desired extent of labeling can be tuned by varying themolar ratio of Sulfo-NHS to GVs and by changing the incubation time aswill be understood by a skilled person. Additional, chemical moietiesincluding polymers (e.g. polyethylene glycol), fluorophores and smallmolecules (e.g. biotin) which can be conjugated methods identifiable.Biotinylated GVs can subsequently react with streptavidin or avidinatedantibodies[20]. Either dialysis or buoyancy purification can be used toseparate the labeled GVs from excess reactants.

In some embodiments methods to functionalize GVs can be performed bygenetically engineering a GvpC protein of the GV shell to include one ormore protein tags.

The term “tag” as used herein means protein tags comprising peptidesequences introduced onto a recombinant protein. Tags can be removableby chemical agents or by enzymatic means, such as proteolysis orsplicing. Tags can be attached to proteins for various purposes:Affinity tags are appended to proteins so that they can be purified fromtheir crude biological source using an affinity technique. These includechitin binding protein (CBP), and the poly(His) tag. The poly(His) tagis a widely-used protein tag; it binds to metal matrices. Chromatographytags can be used to alter chromatographic properties of the protein toafford different resolution across a particular separation technique.Often, these consist of polyanionic amino acids, such as FLAG-tag.Epitope tags are short peptide sequences which are chosen becausehigh-affinity antibodies can be reliably produced in many differentspecies. These are usually derived from viral genes, which explain theirhigh immunoreactivity. Epitope tags include V5-tag, Myc-tag, HA-tag andNE-tag. These tags are particularly useful for western blotting,immunofluorescence and immunoprecipitation experiments, although theyalso find use in antibody purification. Protein tags can allow specificenzymatic modification (such as biotinylation by biotin ligase) orchemical modification (such as reaction with FlAsH-EDT2 for fluorescenceimaging). Tags can be combined, in order to connect proteins to multipleother components. However, with the addition of each tag comes the riskthat the native function of the protein may be abolished or compromisedby interactions with the tag. Therefore, after purification, tags aresometimes removed by specific proteolysis (e.g. by TEV protease,Thrombin, Factor Xa or Enteropeptidase).

Exemplary tags comprise the following, among others known to personsskilled in the art: Peptide tags, such as: AviTag, a peptide allowingbiotinylation by the enzyme BirA and so the protein can be isolated bystreptavidin (GLNDIFEAQKIEWHE (SEQ ID NO:11)); Calmodulin-tag, a peptidethat can be bound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL(SEQ ID NO:12)); polyglutamate tag, a peptide binding efficiently toanion-exchange resin such as Mono-Q (EEEEEE (SEQ ID NO:13)); E-tag, apeptide recognized by an antibody (GAPVPYPDPLEPR (SEQ ID NO:14));FLAG-tag, a peptide recognized by an antibody (DYKDDDDK (SEQ ID NO:15));HA-tag, a peptide from hemagglutinin recognized by an antibody(YPYDVPDYA (SEQ ID NO:16)); His-tag, typically 5-10 histidines that canbe bound by a nickel or cobalt chelate (HHHHHH (SEQ ID NO:17)); Myc-tag,a peptide derived from c-myc recognized by an antibody (EQKLISEEDL (SEQID NO:18)); NE-tag, a novel 18-amino-acid synthetic peptide(TKENPRSNQEESYDDNES (SEQ ID NO:19)) recognized by a monoclonal IgG1antibody, which is useful in a wide spectrum of applications includingWestern blotting, ELISA, flow cytometry, immunocytochemistry,immunoprecipitation, and affinity purification of recombinant proteins;S-tag, a peptide derived from Ribonuclease A (KETAAAKFERQHMDS (SEQ IDNO:20)); SBP-tag, a peptide which binds to streptavidin(MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO:21)); Softag 1, formammalian expression (SLAELLNAGLGGS (SEQ ID NO:22)); Softag 3, forprokaryotic expression (TQDPSRVG (SEQ ID NO:23)); Strep-tag, a peptidewhich binds to streptavidin or the modified streptavidin calledstreptactin (Strep-tag II: WSHPQFEK (SEQ ID NO:24)); TC tag, atetracysteine tag that is recognized by FlAsH and ReAsH biarsenicalcompounds (CCPGCC (SEQ ID NO:25)); V5 tag, a peptide recognized by anantibody (GKPIPNPLLGLDST (SEQ ID NO:26)); VSV-tag, a peptide recognizedby an antibody (YTDIEMNRLGK (SEQ ID NO:27)); Xpress tag (DLYDDDDK (SEQID NO:28)); Covalent peptide tags such as: Isopeptag, a peptide whichbinds covalently to pilin-C protein (TDKDMTITFTNKKDAE (SEQ ID NO:29));SpyTag, a peptide which binds covalently to SpyCatcher protein(AHIVMVDAYKPTK (SEQ ID NO:30)); SnoopTag, a peptide which bindscovalently to SnoopCatcher protein (KLGDIEFIKVNK (SEQ ID NO:31)). Inembodiments described herein, any of the tags of SEQ ID NO:11-31, andother tags known to those skilled in the art, can comprise one or moreamino acid substitutions, insertions, or deletions that do not alter thefunction of the tag, and can further comprise one or more additionalamino acids, up to a maximum tag length of 100 amino acids

In some embodiments, the protein tag can be a polyhistidine tag. Apolyhistidine-tag is an amino acid motif in proteins that typicallyconsists of six histidine (His) residues typically, often at the N- orC-terminus of the protein. It is also known as hexa histidine-tag,6×His-tag, His6 tag and by the trademarked name His-tag (registered byEMD Biosciences). The total number of histidine residues can vary in thetag. N- or C-terminal his-tags can also be followed or preceded,respectively, by a suitable amino acid sequence that facilitates aremoval of the polyhistidine-tag using endopeptidases. This extrasequence is not necessary if exopeptidases are used to remove N-terminalHis-tags (e.g., Qiagen TAGZyme). Polyhistidine-tagging can be used todetect protein-protein interactions in the same way as a pull-downassay. Fluorescent hexahistiadine CyDye tags are also available. Theseuse Nickel covalent coordination to EDTA groups attached to fluorophoresin order to create dyes that attach to the polyhistidine tag. Thistechnique has been shown to be effective for following protein migrationand trafficking. This technique may also be effective in order tomeasure distance via Fluorescent Resonance Energy Transfer.

In embodiments described herein a GvpC or a variant gvpC can beengineered to attach a tag fused to or inserted into an N-terminalregion, a C-terminal region of a gvpC or a variant gvpC. In someembodiments, a tag that can be used for affinity purification of theengineered gvpC, such as a His-tag. In some embodiments, the tagcomprises one or more functional groups that can be used to alter thesurface charge of a GV, such as a lysine-rich protein (LRP). In someembodiments, a tag comprises a moiety that can be used for targeting aGV to a cell, such as a receptor-targeting peptide RGD, which bindseffectively to a wide range of integrins. In some embodiments, a tagcomprises a functionalized moiety that can be used to increase ordecrease uptake of GVs by macrophages, such as a CD47 or an R8,respectively. In some embodiments, a tag can comprise a functionalizedmoiety that can be used for modular approaches in which the GV surfacecan be specifically covalently conjugated to other recombinant proteins,such as a SpyTag-SpyCatcher.

In some embodiments, engineering of a GvpC to attach one or more tagscan be performed with or without substantially alter the criticalcollapse pressure of the base GvpC.

For example in some embodiments described herein, a GvpC protein of a GVcan be engineered to attach one or more protein tags or polypeptide tagswhile optionally substantially altering the acoustic collapse pressureof a GV shell comprising the engineered GvpC as compared to a GV shellof a same non-engineered GvpC.

The term “substantially alter” or “substantially decrease” as usedherein means a decrease of more than 10% in acoustic collapse pressure,preferably more than 20% in acoustic collapse pressure.

In some embodiments described herein, an engineered GvpC protein cancomprise one or more protein tags or polypeptide tags. In embodimentsdescribed herein, appending functional residues comprising one or morepolypeptide tags or protein tags to the N-terminus or the C-terminus ofGvpCWT can reduce collapse pressure depending on the length and exactproperties of the amino acid sequence.

In particular, in some embodiments, engineering of a GvpC can be furtherengineered to attach one or more tags up to the C-terminus withoutsubstantially alter the critical collapse pressure as compared todeleting the N- and/or C-terminal regions. In some embodiments, smalltags such as RGD and RDG do not substantially alter the collapsepressure value. In some embodiments, tags comprising longer sequencessuch as LRP (100 residues) decrease acoustic collapse pressure to agreater extent. In some embodiments, tags such as those comprising mCD47cause a substantial decrease in acoustic collapse pressure value. Insome embodiments, appending a His-Tag (e.g. 6 His amino acids) to theN-terminus of the wild-type GvpC sequence does not substantially alterthe acoustic collapse pressure value. In some embodiments, appending agvpC with a Spytag-Spycatcher (FIG. 12) is an effective method tofunctionalize GVs with large molecules (greater than 100 amino acids inlength) such as fluorescent proteins, without substantially alteringtheir collapse pressure value.

Addition of a functional moiety comprised in a tag to a gvpC or avariant gvpC can be obtained through different approaches identifiableby a skilled person.

For example, in some embodiments, an addition of a tag to a gvpC orvariant gvpC can be performed at a protein level by first providing thegvpC protein or variant gvpC protein and the protein tag and thenperforming the insertion into a N-terminal or aC-terminal region bybreaking a peptide bond between two adjacent amino acids of the gvpC orvariant gvpC N-terminal region of C-terminal region and then forming newpeptide bonds between the gvpC or variant gvpC and the protein tag, asdescribed above. For example, the gvpC or variant gvpC can be digestedwith a protease to break a peptide bond between two adjacent amino acidsin the gvpC or variant gvpC N-terminal region or C-terminal region,followed by insertion of the protein tag between the previously adjacentamino acids of the N-terminal region or C-terminal region, for exampleusing native chemical ligation methods known to those skilled in the art[21]. In other embodiments, a protein tag can be fused to a C-terminusor an N-terminus of a gvPC protein or a variant gvpC protein usingnative chemical ligation methods known in the art.

In some embodiments, a tagged gvpC or variant gvpC functionalized with aprotein tag inserted or fused to the N- or C-terminus can be synthesizedas single protein by design. Proteins can be synthesized usingbiosynthetic methods, such as cell-based methods or cell-free methodsknown to those skilled in the art. Protein biosynthesis can be performedby translation of DNA or RNA polynucleotides encoding the protein. Thus,protein biosynthesis can be performed by providing cell-based orcell-free protein translation systems with DNA or RNA polynucleotidesencoding the protein. For example, protein biosynthesis can be performedin cells transfected with in vitro transcribed RNA encoding the protein.Proteins can also be produced by liquid-phase or solid-phase chemicalprotein synthetic methods known to those skilled in the art [22].

In some embodiments, insertion or terminus fusion of a protein tag to agvpC or a variant gvpC can be performed at a polynucleotide levelthrough to an in-frame insertion of a protein tag-coding polynucleotidein between two codons in an N- or C-terminal region of a gvpC or avariant gvpC. An in-frame insertion can be performed in several steps,by first providing the gvpC- or variant gvpC-coding and the proteintag-coding polynucleotides and performing the insertion by breaking abond (typically a phosphodiester bond) between two adjacent nucleotidebases of the first polynucleotide and then forming new bonds between thegvpC-coding polynucleotide and the protein tag-coding polynucleotide.For example, the gvpC coding polynucleotide can be digested with one ormore restriction endonucleases and then the protein tag-codingpolynucleotide inserted by ligation (e.g., using T7 DNA ligase) intocompatible site(s) allowing formation of phosphodiester bonds betweenthe first and second polynucleotide bases. Compatible DNA ligation sitescan be “sticky” ends, digested with restriction endonuclease producingan overhang (e.g. EcoRI), or can be “blunt ends” with no overhang, aswould be understood by those skilled in the art. A fusion of apolynucleotide encoding a tag can also be ligated to an N- or C-terminusof a gvpC or a variant gvpC polynucleotide by ligation (e.g., using T7DNA ligase) into compatible site(s).

In some embodiments, the gvpC- or variant gvpC-coding and the proteintag-coding polynucleotides can be provided within a singlepolynucleotide by design. For example, a tag can be added by insertingthe polynucleotide encoding a protein of interest in a plasmid or vectorthat has the tag ready to fuse at the N-terminus or C-terminus. The tagcan be added using PCR primers encoding the tag; using PCR the tag canbe fused to the N-terminus or C-terminus of the protein-codingpolynucleotide, or can be inserted at an internal location, usinginternal epitope tagging [23], among other methods known to thoseskilled in the art. Other methods such as overlap extension PCR andinfusion HD cloning can be used to insert a tag at a site between theN-terminus and C-terminus of a protein-coding polynucleotide (seeExamples). Optionally, a polynucleotide encoding a ‘linker’ (such as asequence encoding a short polypeptide or protein sequence, e.g.,gly-gly-gly or gly-ser-gly can be placed between the protein of interestand the tag; this can be useful to prevent the tag from affecting theactivity of the protein being tagged.

The choice of the location where a tag is added to a protein sequencedepends mainly on the structural and functional features of a proteinand the intended downstream methods employing the use of the tag.

In embodiments herein described, the insertion location of a protein tagin a genetically engineered gvpC or variant gvpC is performed atinsertion position selected to have the tag presented on the externalsurface-exposed position of the gvpC or variant gvpC withoutcompromising the function of the gvpC or variant gvpC.

In some embodiments, the GVPS and variants thereof can be used as acontrast agent in the method to provide an ultrasound imaging of atarget site. In some embodiments, the GVPS and variants thereof can beused as a contrast agent in the multiplexed ultrasound imaging methodsherein described. In particular, a combination of GVPS and/or variantsthereof can be used in a contrast agent, each exhibiting a differentacoustic collapse profile with progressively decreased midpoint collapsepressure values. In some cases, the percentage difference between themidpoint collapse pressure values of any given two GVPN types in thecontrast agent is at least twenty percent.

In some embodiments, one or more of the GVs herein described can becomprised in a composition together with a suitable vehicle. The term“vehicle” as used herein indicates any of various media acting usuallyas solvents, carriers, binders or diluents for the multi-ligand captureagents that are comprised in the composition as an active ingredient. Inparticular, the composition including the GVs can be used in one of themethods or systems herein described

In some embodiments, one or more of the GVs herein described can becomprised in a contrast agent in the sense of the disclosure, thecontrast agent comprising a plurality of the GVs (inclusive ofengineered GVs) and/or GvpC variants herein described. The term“contrast agent” refers to an agent (material) in aqueous media,including water, saline, buffer, liquid media, configured to increasecontrast in ultrasound imaging methods. By an increase in contrast, itis meant that the differences in image intensity between adjacenttissues visualized by a ultrasound imaging method are enhanced. Forinstance, differences in image intensity can be enhanced with the useone or more sets of imaging parameters.

The contrast agent can be provided in any pharmaceutically and/orphysiologically suitable liquid or buffer known in the art. For example,the contrast agent can be contained in water, physiological saline,balanced salt solutions, buffers, aqueous dextrose, glycerol or thelike. In certain embodiments, the contrast agent can be combined withagents that can stabilize and/or enhance delivery of the contrast agentto the target site. For example, the contrast agent can be administeredwith detergents, wetting agents, emulsifying agents, dispersing agentsor preservatives.

In certain embodiments, two or more gas vesicle types are combined in amixture for multiplexed imaging, wherein the two or more GV types havedistinct acoustic collapse profiles and different biodistribution ortargeting properties, such that their location in the imaged specimenprovides information about two or more different aspects of thespecimen, such that the unmixed ultrasound images acquired afteradministering this mixture contains information about the two or moredifferent aspects of the specimen. Different aspects of the specimen mayinclude different molecular or cellular targets to which the GVs bind,different vascularization patterns through which GVs flow incirculation, different levels of cellular metabolism leading to uptakeor destruction of GVs, etc. These mixtures are supplied together withthe acoustic collapse profiles or each component of the mixture and theMIAPs that should be used to acquire multiplexed images.

In certain embodiments, the gas vesicles contained in the contrast agentare bacterially-derived, gas vesicles formed by bacteria, such asphotosynthetic bacteria (e.g., cyanobacteria), or archaea-derived gasvesicles formed by archaea (e.g., halobacteria). In other embodiments,the gas vesicles contained in the contrast agent are geneticallyengineered GVs by genetically engineering the bacterially-derived gasvesicles or archaea-derived gas vesicles herein described.

As mentioned above, the GVs (inclusive of native and variant GVs) and/orGvpC variants herein described can be provided as a part of systems toperform any of the above mentioned methods. The systems can be providedin the form of kits of parts. In a kit of parts, one or more GVs and/orGvpC variants and other reagents to perform the method are comprised inthe kit independently. The GVs and/or GvpC variants can be included inone or more compositions, and each GV and/or GvpC variant is in acomposition together with a suitable vehicle.

In particular, the components of the kit can be provided, with suitableinstructions and other necessary reagents, in order to perform themethods here disclosed. The kit will normally contain the compositionsin separate containers. Instructions, for example written or audioinstructions, on paper or electronic support such as tapes or CD-ROMs,for carrying out the assay, will usually be included in the kit. The kitcan also contain, depending on the particular method used, otherpackaged reagents and materials (such as. wash buffers and the like).

Further details concerning the engineered GVs, and systems and methodsof the present disclosure will become more apparent hereinafter from thefollowing detailed disclosure of examples by way of illustration onlywith reference to an experimental section.

EXAMPLES

The engineered GVs and related systems and methods herein disclosed arefurther illustrated in the following examples, which are provided by wayof illustration and are not intended to be limiting.

In particular, the following examples illustrate exemplary methods andprotocols for preparing exemplary gas vesicles protein structure fromAna, Halo and engineered E. Coli, and related characterizing testing anduse these structure for ultrasound imaging in vivo and in vitro. Aperson skilled in the art will appreciate the applicability and thenecessary modifications to adapt the features described in detail in thepresent section, to additional gas vesicle protein structure and relatedmethods and systems according to embodiments of the present disclosure.The following materials and methods were used in the experimentsillustrated in the Examples.

Transmission Electron Microscopy

GV samples were diluted to O.D_(PS,500)˜0.2 in 10 mM HEPES buffercontaining 150 mM NaCl (pH 8) and spotted on Formvar/Carbon 200 meshgrids (Ted Pella, Redding, Calif.) that were rendered hydrophilic byglow discharging (Emitek K100X). GV samples were negatively stainedusing 2% Uranyl Acetate. Images were acquired using the Tecnai T12 LaB6120 kV TEM equipped with a Gatan Ultrascan 2 k×2 k CCD and ‘Leginon’automated data collection software suite.

Pressurized Absorbance Spectroscopy

GV samples were diluted to O.D_(PS,500)˜0.2 and loaded onto aflow-through, 1 cm path-length quartz cuvette (Hellma Analytics,Plainview, N.Y.) that was connected to a N₂ cylinder through a pressurecontroller (Alicat Scientific, Tucson, Ariz.). The pressure wasincreased stepwise in 20 kPa increments up to 1 MPa and the O.D_(PS,500)at each step was measured using a spectrophotometer (EcoVis,OceanOptics, Winter Park, Fla.). Fully collapsed GV sample was used asthe blank.

In Vitro Ultrasound Imaging

Imaging phantoms were prepared from 1% agarose in PBS. Two timesconcentrated GV samples were mixed 1:1 with melted 1% agarose at 50° C.,and 100 μL of the mixture was quickly loaded into the phantom wells.Imaging was performed using a Verasonics Vantage programmable ultrasoundscanning system. The L11-4v or L22-14v 128-element linear arraytransducers (Verasonics, Kirkland, Wash.) were used for imageacquisition, with a pitch of 0.3 mm or 0.1 mm and elevation focus of15-20 mm or 6 mm respectively. The phantom was placed on a custom 3-Dprinted holder and the transducer was mounted on a computer-controlled3-dimensional translating stage (Velmex, Inc., Bloomfield, N.Y.). Duringimaging, the transducer was immersed in PBS at an elevation thatpositioned the focal zone of the ultrasound beam at the center of thesample well. All images were acquired using a conventional B-modesequence with 128 ray lines.

The acoustic multiplexing and collapse spectrum measurements wereobtained by using GV samples at a final OD of 1 and a transmit frequencyof 6.25 MHz on the L11-4v, with a 4 cycle pulse and transmit focus of 20mm, F-number 2 and persistence 90. The images were acquired at atransmit voltage of 1.6 V. To collapse GVs, acoustic pressure wasdelivered to the specimen by lowering the F number to 0.1 and ramping upthe voltage gradually. At each collapse step, the transducer wastranslated in the y and z planes to ensure homogenous GV collapse overthe entire well.

Non-linear imaging experiments were performed using the L11-4vtransducer with a transmit frequency of 4.46 MHz and receive filteringusing a 2 MHz band pass around 4.46 MHz and 8.92 MHz for the fundamentaland second harmonic signals, respectively. GV samples at OD 2.5 wereimaged at 2.5 V and F-number 3 using a 3 cycle pulse and a persistenceof 90.

In Vivo Ultrasound Imaging

Intravenously injected gas vesicles were imaged in 5-7 weeks old femaleSCID mice using the L11-4v transducer. To be consistent with in vitroexperiments, a transmit frequency of 4.46 MHz and reception frequenciesof 4.46 MHz and 8.92 MHz were used for the fundamental and non-linearimaging respectively. Imaging was done at 2.5 V using a 3 cycle pulse atan F-number 3 and persistence of 20. The mice were maintained underisofluorane anesthesia on a heated imaging platform. Images wereacquired at a rate of 16 frames/sec for ˜50 s. A 50 μL volume of gasvesicles at OD 23.5 in PBS was infused ˜5 s after the start of theexperiment at a flow rate of 0.3 ml min⁻¹. Between sample injections, a10 s high-power burst from the transducer was used to completelycollapse any residual GVs in circulation.

Image Analysis

MATLAB and ImageJ (NIH, Bethesda, Md.) were used to process in vitro andin vivo ultrasound data. Regions of interest (ROIs) were manuallydefined so as to capture signals from the entire sample well or the IVC.ROI dimensions were preserved between different GV samples and the meanintensity per pixel calculated using all pixels within the ROI.Quantification of in vitro harmonic and fundamental GV signals wasperformed by subtraction of the post-collapse images from thepre-collapse images. In vivo IVC signals were analyzed for all acquiredframes over the 50 s imaging window and smoothed infusion time-coursecurves were generated using locally weighted scatterplot smoothing. Areaunder the curve (AUC) values were obtained from the raw data normalizedto the pre-infusion baseline. Acoustic spectral unmixing was performedusing MATLAB after applying a spectral averaging filter with a kernelsize of [20 20] pixels to reduce out-of-well noise. Pseudocolorassignments and merging of spectrally unmixed images were performedusing ImageJ (greyscale converted representations of color maps areshown next to the images in FIG. 4 Panel g).

Zeta Potential Measurements

Zeta potential of GVs with WT-GvpC and GvpC-LRP were measured usingBrookhaven Instruments Corporation Zeta-PALS instrument (Hotsville,N.Y.). 40 μL of GVs (in PBS) were added to 1.5 mL of double distilledwater at a final concentration of 35 pM and conductance of 1 mS.Electrodes were placed in the cuvette with the samples and average zetapotential for each run was determined from 10 measurements.

In Vitro Characterization of Functionalized GVs

Alexa-488 succinimidyl ester fluorescent dye (Invitrogen, Carlsbad,Calif.) was reacted with GVs in PBS for 2 hours at 10, 000:1 molarexcess of dye to GVs. Excess succimidyl ester was quenched with 10 mMTris. Fluorescently-labeled GVs were purified using dialysis againstPBS. Cells were seeded on 22×22 mm coverglass and cultured for 24 hoursprior to the start of the experiments. Due to the buoyant nature of GVs,in vitro characterization was carried out using modified 6-well platesthat contain 3 pegs to enable inverted cell growth (facing down). Forreceptor (α_(v)β₃) targeting experiments, 16 μL of fluorescently-labeledGVs (GvpC_(WT), GvpC_(WT)-RGD, and GvpC_(WT)-RDG) at 1.2 nM were addedto U87 cells (ATCC Manassas, Va.) and incubated for 24 hrs. To testphagocytic uptake using GvpC_(WT), GvpC_(WT)-mCD47, and GvpC_(WT)-R8, 8μL of fluorescently-labeled GVs at 1.2 nM were added to RAW 264.7 cells(ATCC). After the allotted GV incubation, cells were washed 3× with PBS,fixed with 4% paraformaldehyde, and mounted with DAPI containingmounting media. Confocal fluorescence images were acquired usinginverted Zeiss LSM 710 NLO (Thornwood, N.Y.) using a 20× objective.

SpyTag—SpyCatcher Functionalization of Ana GVs

SpyTag-Ana GVs were prepared using the re-addition protocol describedabove. SpyCatcher-mNeonGreen (SC-mNG) was expressed and purified fromBL21 E. coli using non-denaturing Ni-NTA purification. ST-GVs (OD: 5-10)were incubated with SC-mNG at a 2× molar excess of SpyCatcher:SpyTag inPBS for 1 h at room temperature. GVs were spun at 300 g for 4 hourstwice in order to remove excess unbound protein; the supernatantcontaining GVs was resuspended in fresh PBS.

ST-GV (±SC-mNG) and WT-GV (+SC-mNG) samples were prepared in a 1%agarose phantom at final OD 2.5 and imaged with the Verasonics L22-14Vtransducer at 19 MHz, 5.0V and F-Number 3 with a persistence of 90. Theagarose phantom was also imaged through the green channel of a BioRadChemidoc MP system (Hercules, Calif.). The fluorescence intensity of theST-GV (±SC-mNG) and WT-GV (+SC-mNG) samples was determined by firstcollapsing the samples and then measuring fluorescence intensity (ex 506nm, em 550 nm) in a Molecular Devices SpectraMax M5 plate reader(Sunnyvale, Calif.).

Example 1: Overview of Exemplary Procedure to Express and Purify GVs

A detailed exemplary optimized protocol is illustrated to express andpurify GVs and quantify, optionally functionalize and geneticallyengineer them, characterize their size and shape, and use them ascontrast agents for non-invasive imaging applications.

The procedure begins with isolation of GVs from cultures of Ana andHalo, or from E. coli expressing a heterologous GV gene cluster fromBacillus megaterium (Mega)[18]. This results in three structurepopulations with distinct properties (see Example 2 and Table 1).

These GVs are then quantified and characterized using pressure-dependentspectroscopy, dynamic light scattering (DLS) and transmission electronmicroscopy (TEM).

Protocols for modification and functionalization using genetic andchemical approaches are provided as an added option based on the desiredend application. Finally, the GVs are imaged in vitro and in vivo usingultrasound and in vitro using HyperCEST MRI.

Example 2: Experimental Design

Key experimental parameters for each stage of the protocol are taken inconsideration in performing an experimental design to produce a GV forultrasound imaging. One important initial consideration is the choice ofGV-producing species. Different of GV-producing microorganisms provideGVs with different characteristics which affect the related use ascontrast in ultrasound imaging.

Although this protocol presents methods for producing three differenttypes of GVs—Ana, Halo and Mega—one of these types may be mostappropriate for a given application (see Table 1).

TABLE 1 Characteristics of different types of GVs Ana GV Halo GV Mega GVHost/origin Anabaena Halobacteria Heterologous flosaquae salinarumexpression of a gene cluster from Bacillus megaterium in E. coli. ShapeCylindrical Spindle Cylindrical Resistance to Medium Low High pressure-(can be tuned) induced collapse Ultrasound contrast High High LowNonlinear after Nonlinear engineering Stability in High Low High Xe-MRIphantoms Ease of genetic High Low Not established modification

For example, for ultrasound, unmodified Halo GVs can be used directly inultrasound imaging to obtain non-linear signals [20, 24]. Ana GVs are asystem of choice if one wishes to genetically tune the properties of GVsfor multiplexing, multimodal imaging and targeting applications [1, 25].Mega GVs produce lower echogenicity under ultrasound compared to Ana andHalo GVs, but have a higher critical collapse pressure that can makethem useful for multiplexing.

Halo GVs produce non-linear ultrasound contrast immediately afterpurification, while Ana GVs require a chemical treatment. With regard toXe-MRI, Ana and Mega GVs are more stable under pressure and during thebubbling of hyperpolarized xenon compared to Halo GVs[26]. All threespecies have a unique chemical shift in Xe-MRI, allowing multiplexing

Example 3: Production of GVs from Ana and Halo or HeterologouslyExpressing E Coli

GVs are obtained from Ana, Halo or heterologously-expressing E. coli.Ana is cultured in low-salinity medium supplemented with trace metalsand buffering agents, while Halo is cultured in high salinity medium forGV production.

Ana and Halo cultures natively produce ample GVs after a few weeks ofgrowth[1, 20, 25]. Ana cultures additionally require a controlledgaseous environment and illumination for optimal growth. As understoodby those skilled in the art, typically, a freshly inoculated culture ofcells may require several rounds (typically 2-3) of subculture to becomestrongly proliferative. The confluent culture of microbes can then betransferred to a separatory funnel that is left undisturbed for up to aweek to allow the buoyant cells producing GVs to float to the top andseparate from media. Buoyant cells are then lysed using hyper-osmoticshock for Ana and hypo-osmotic shock for Halo. Subsequently,centrifugally assisted floatation can be used to isolate GVs from thecell lysate to yield a concentrated, milky-white solution of GVs in thebuffer of choice[1, 20, 25]. Heterologous production of Mega GVs in E.coli can be accomplished by expression from a plasmid encoding a Mega GVgene, followed by detergent-mediated lysis[26].

The procedures leading from inoculation of GV-producing microbes toharvesting and purification is summarized in the Table 9, along withimportant parameters that affect processing time, yield and quality.

TABLE 9 Experimental Parameters for GV Production, Purification andStorage Procedure Design Parameters Inoculation of starter Type ofculture (suspension vs. solid), culture amount of inoculum, total volumeof culture Growth of starter Temperature, rotation speed, duration,illumination culture Sub-culturing Number of flasks, volume of cultureand media Harvesting of GVs Composition of lysis buffer and duration oflysis, concentration of cells Purification Selection of centrifugationspeed, type of rotor, tube and syringe needle Storage Storagetemperature, buffer and type of vial/tube

Growth conditions are chosen to facilitate optimal proliferation of eachhost strain and GV expression. As understood by those skilled in theart, one variable to keep track of is pressure, since GVs collapseirreversibly at hydrostatic pressures of 50 to 800 kPa, depending onspecies[3]. For example, the cultures should be grown under mildagitation, as excessive shaking can lead to GV collapse. Duringcentrifugation steps, the hydrostatic pressure generated for aparticular g-force on the liquid column of GVs can be calculated, toensure that it is well below the GV critical collapse pressure.Long-term storage of purified GV stocks should preferably be done inscrew-top vials, as micro-centrifuge tubes with snap-lock caps may causeGV collapse due to pressurization of the sample while opening or closingthe tube.

Example 4: Expression and Purification of Ana GVs

Anabaena floc-aquae (Ana) was cultured in Gorham's media supplementedwith BG-11 solution (Sigma, St. Louis, Mo.) and 10 mM NaHCO₃ at 25° C.,100 rpm shaking and 1% CO₂ under a 14 h light cycle and 10 h dark cycle.Once confluency was reached, the cultures were transferred to sterileseparating funnels and the buoyant cells were allowed to float to thetop and separate from the spent media over a 48 h period. Ana GVs wereharvested by hypertonic lysis of the buoyant cells with 500 mM sorbitoland 10% Solulyse (Genlantis, San Diego, Calif.). Purification was doneby repeated centrifugally assisted floatation followed by resuspensionin 1×PBS (Corning, Union City, Calif.). GV concentration was determinedby pressure-sensitive OD measurements at 500 nm (OD_(PS,500)).Pre-collapsed GVs prepared by application of hydrostatic pressure in acapped syringe were used as the blank.

Example 5: Expression and Purification of Halo GVs

(i) An exemplary method for growing Halo cultures for GV productionfollows. Aliquot Carolina growth medium in to an autoclaved flask understerile conditions. (ii) Inoculate Halo cultures under sterileconditions using one of the following methods. (1) scrape a small amountof pink culture from an agar plate to add to the flask as inoculum. (2)Use one to two brine crystals containing Halo for inoculation. (3)Inoculate from a healthy pink liquid starter culture into 250 mL offresh growth medium. (iii) Grow the culture in an incubator at 42° C.with 100 rpm shaking for ˜2 weeks or until the inoculated culturebecomes confluent.

(iv) An exemplary method for harvesting Halo GVs follows: Gently pourthe culture from the flask into a separatory funnel (pre-sterilized withstopcork in place). Allow the culture to remain undisturbed until avisible ring is formed at the top. This typically takes 4-6 days. (v)Remove as much of the spent media as possible by opening the stopcork,retaining only the buoyant layer of milky-pink cells for lysis. (vi)Using equal volume of TMC lysis buffer (pH 7.5), gently wash the cellsstuck on the sides of the funnel and retrieve the cells. The volume ofTMC buffer used might be varied depending on the cell density to achieveefficient hypo-osmotic lysis.

(vii) An exemplary method for isolation and purification of Halo GVsfrom lysate follows: Aliquot ˜1.6 mL of cells in 2 mL tubes and spin ina microcentrifuge at 300 g for 4 hours at 8° C. It is critical to closethe tubes gently; the pressure wave caused from snapping the lid willcollapse a large number of Halo GVs. (viii) At the top of the tube, amixed layer of Halo GVs (white) and unlysed Halo cells (milky-pink) willbe visible. Using a blunt end 18.5 or 21.5 G needle, aspirate the pelletat the bottom of the tubes as well as the pink cell lysate. Take care tolimit the amount of floating Halo cells and Halo GVs (white) that areaspirated in to the syringe. (ix) Transfer the GVs and unlysed Halocells to fresh tubes and bring to 1.6 mL with 1×PBS. Centrifuge tubes at300 g for 4 hours at 8° C. (x) Repeat steps viii and ix. After eachstep, the amount of milky-pink buoyant cells will reduce and white HaloGVs will increase. Continue with centrifugally-assisted floatation untilall the cells have lysed and there is no evidence of pink cell lysate inthe subnatant. (xi) Resuspend the purified GVs in PBS and aliquot themilky white GV solution into screw top vials or microcentrifuge tubes.The aliquoted Halo GVs can be stored for up to one year at 4° C. Avoidfreezing and subjecting the tube to mechanical shocks, such as droppingto the ground or snapping the cap, as this may collapse the GVs.

Example 6: Expression and Purification of Mega GVs

An exemplary protocol for heterologous expression of Mega GVs in E. colifollows: Transform 50 μL chemically competent Rosetta™ 2 (DE3) pLysScells using >1 ng of pST39 plasmid containing the pNL29 Mega GV genecluster [26] by mixing the two components in a 1.5 mL tube andincubating on ice for 30 minutes. Heat shock the tube in a 42° C. waterbath for 45 seconds, and put the tube back on ice for a minute. Add 500μL of SOC outgrowth medium and incubate in a shaker at 37° C. and 250rpm for 1 hour. (ii) Prepare 3 mL of LB broth containing 1× Ampicillin(100 μg/mL), 1× Chloramphenicol (25 μg/mL) and 1% (wt/vol) glucose in aglass culture tube. Resuspend 300 μL of the transformed E. coli in thebroth. Grow the culture in a shaker-incubator at 37° C. and 250 rpmuntil OD₆₀₀ reaches 0.4-0.6. Make 100 μL aliquots of the culture insterile tubes, and mix with 100 μL of 50% sterile glycerol. Freeze thetubes at −80° C. as E. coli glycerol stocks. The glycerol stocks can bestored at −80° C. and used for up to 3 months. Note that the GV yield isreduced when using frozen stocks, so fresh overnight transformations arepreferred. (iii) Resuspend a tube of the aliquoted glycerol stock in 3mL LB broth containing 1× Ampicillin, 1× Chloramphenicol and 1% (wt/vol)glucose. Grow the E. coli culture to saturation (OD₆₀₀>4). For freshtransformations, aliquot 500 uL of the transformed E. coli from step (i)into 5 mL of LB broth containing 1× Ampicillin, 1× Chloramphenicol and1% (wt/vol) glucose. Grow overnight until the culture reaches saturation(˜16 hours). (iv) Prepare 100 mL LB broth containing 1× Ampicillin, 1×Chloramphenicol and 0.2% (wt/vol) glucose, and inoculate 1 mL of thesaturated E. coli culture into the broth. Grow at 37° C. for ˜2 hoursuntil OD₆₀₀ reaches 0.4 to 0.6. Induce the culture by adding 20 μM IPTG(final concentration), and grow at 30° C. for an additional 16-24 hours.

(v) An exemplary protocol for harvesting and purifying Mega GVs from E.coli cultures follows: Split the culture equally into three 50 ml Falcontubes and spin for 1 hour at 500 g and 25° C. Avoid higher speedsbecause they may cause collapse of GVs. (vi) Insert a 10 mL syringe withneedle to >1 cm below the surface of the solution and withdraw the clearliquid component of the solution. Withdraw the liquid slowly to preservethe thin layer of cells floating at the top of the solution, as well asthe pellet at the bottom, both of which contain Mega GVs. (vii) To lysethe cells, add 4 ml SoluLyse-Tris reagent per 50 ml of E. coli culture,250 μg/ml lysozyme and 10 μg/ml DNAseI. Rotate the tubes for 10 minutesat room temperature and then aliquot 1.5 mL of the solution to 2 mLtubes. Spin samples for 4 hours at 800 g and 8° C. Mix the floating GVlayer gently with supernatant and transfer to a clean tube. (viii) Spinthe samples for 4 h at 800 g. Use a 3 mL syringe to remove the bottomfraction, which sometimes includes a small pellet. Gently resuspend GVsin 1 mL of PBS. Repeat the spin and wash steps 3 times. GVs aresusceptible to desiccation; resuspend GVs immediately after withdrawingthe liquid. (ix) Mega GVs are natively clustered. To uncluster them,GV-containing solution is mixed with 10 M urea in a 2:3 ratio to achieve6 M final urea concentration, and the resulting solution is gentlyrotated for 30 min. (x) Dialyze GVs overnight in 6-8 kDa MWCO tubingagainst 4 L of PBS. This step can be omitted for experiments with nostringent requirements for buffer conditions. A white layer ofunclustered GVs is at the top of the liquid phase after buoyancypurification, as well as a re-suspended milky-white solution of Mega GVsin the PBS. Mega GVs can be stored for up to one year at 4° C. Avoidfreezing and subjecting the tube to mechanical shocks, such as droppingto the ground or snapping the cap, as this may collapse the GVs.

Example 7: Quantification and Characterization of Gas Vesicles ProteinStructures by Pressure Dependent Spectroscopy

As understood by those skilled in the art, GVs can be quantified andcharacterized using techniques such as pressure-dependent spectroscopy,dynamic light scattering (DLS) and transmission electron microscopy(TEM), among others.

Purified GVs resuspended in a buffer (e.g. phosphate buffered saline,PBS) can be quantified by measuring the optical density at 500 nm, orOD₅₀₀, since GVs scatter visible light. Collapsed GVs (in the samebuffer), which do not scatter light, are typically used as the blankcontrol for measurements, yielding a pressure-sensitive OD reading(OD_(500, ps)). As understood by those skilled in the art, thatclustering of GVs, whether by design or due to functionalization withaggregation-prone moieties can potentially confound OD₅₀₀ measurementsand contribute to errors in calculating concentration from OD₅₀₀.Pressurized absorbance spectroscopy assays GV mechanical strength bymeasuring OD₅₀₀ under increasing hydrostatic pressure using a device asshown in FIG. 15.

The concentration of a solution of gas vesicles can be determined bymeasuring its pressure-sensitive optical density at 500 nm (OD_(500,ps))using a NanoDrop 2000 Spectrophotometer. Load 2 μL of sample on thepedestal for each measurement. Collapsed gas vesicles in the same bufferare used as a blank for measurements. Prepare collapsed GVs bysonication in a water bath until the solution turns completely clear orby manual collapse in a capped syringe. For manual collapse, remove theplunger from a 12 mL Luer-Lock syringe closed with a tip cap and place5-10 μL of gas vesicle solution at the bottom of the syringe. Makingsure that the tip cap is screwed on tight, replace the plunger and pushdown until there is significant resistance. The increase in pressurewill collapse the gas vesicles, turning the milky white solution clear.A shortcut for quick measurements of GV concentration is to blank withthe GV resuspension buffer. For most samples, this will give an ODreading that is very close to that measured when using collapsed GVs asa blank. However, for some samples containing GVs that are fluorescent,the collapsed GVs are used as the blank. As would be understood by thoseskilled in the art, it can be important to ensure that the GVs arehomogenously re-suspended in solution just before measurements. For eachsample, take the average OD_(500,ps) value after multiple measurements(n>=3) to ensure precision and accuracy.

To characterize the purified GVs, the following exemplary procedure canbe used to determine the critical collapse pressure of GVs usingpressurized absorbance spectroscopy. Before acquiring measurements,connect the spectrophotometer to a power supply for 30 m to allow it towarm up. Run the Alicat startup script to initialize the pressurecontroller. Blank the spectrophotometer with a cuvette filled with PBSor GV resuspension buffer and run OceanOptics_startup_FullTrans to savethe data. Establish a zero-transmission baseline with the opaque side ofcuvette blocking the light path using the OceanOptics_startup_NoTransscript. Fill cuvette with intact GV sample (OD_(500nm)=0.2 in PBS) andfasten the cannulae securely. To assist loading, use elongatedgel-loading micropipette tips. Open the N₂ tank valve, pressureregulator, and pressure controller valve. Run the Collapse script tomeasure the OD_(500,ps) under increasing hydrostatic pressure (0 kPa-1.4MPa in 20-kPa increments). Between these measurements, rinse the cuvettewith DI H₂O, 70% ethanol and acetone to ensure that cuvette iscompletely clean and dry before adding the next sample. Aftermeasurements, close gas the valves and turn off the spectrophotometer.

GV protein concentrations can be measured for example using the Pierce660 nm protein assay to obtain relationships between optical density andprotein content for the GV solutions. The protein concentrations to ODrelationships of three types of GVs can be determined, for example asshown in Table 2 below (N=4, 5, 3 for Mega, Ana and Halo GVsrespectively and the errors are in SEM). The molecular weight can bederived from the TEM data, assuming a spindle shape for Halo GVs, acylindrical shape for Mega and Ana GVs, a wall thickness of 18 Å and aprotein density of 1.4 g/mL.

TABLE 2 Quantification and calculation of GV molecular weight and molarconcentration. Ana Halo Mega Protein concentration to 36.6 ± 2.6 13.4 ±2.2 145.5 ± 6.4 OD₅₀₀ ratio ([μg/mL]/OD) Estimated molecular 320 28271.7 weight (MDa) Estimated molar 114 47.3 2,030 protein concentrationto OD₅₀₀ ratio (pM/OD) Estimated gas fraction to 0.000417 0.0001780.000794 OD₅₀₀ ratio (v/v/OD)

Additional techniques to quantify and characterize GVs are exemplifiedin additional examples herein described

Example 8: Quantification and Characterization of Gas Vesicles ProteinStructures by Dynamic Light Scattering

Dynamic light scattering (DLS) can be used to estimate the hydrodynamicsize of GVs for routine non-destructive characterization and qualitycontrol. DLS can be used to assess GV clustering. As would be understoodby those skilled in the art, care should be taken in the interpretationof DLS readings of GVs due to the spherical assumption of theEinstein-Smoluchowski relation and the non-spherical shape of GVs.

An exemplary protocol for preparation of GV specimens for dynamic lightscattering (DLS) is as follows: First, dilute the GV samples toOD_(500,ps)=0.2 in PBS. Next, measure the particle size on ZetaPALSinstrument with an angle of 90° and refractive index 1.33.

Additional protocols would be identifiable by a skilled person uponreading of the present disclosure.

Example 9: Quantification and Characterization of Gas Vesicles ProteinStructures by Transmission Electron Microscopy (TEM)

An exemplary protocol for preparation of GV samples for transmissionelectron-microscopy (TEM) follows: First, buffer-exchange the purifiedGVs in 10 mM HEPES buffer with 150 mM NaCl or an alternativenon-phosphate containing buffer via centrifugally-assisted floatation(same procedure as used for GV isolation) at 8° C. and 300 g. Replacethe subnatant with equal volume 10 mM HEPES buffer with 150 mM NaCl.Repeat 3 times. The aim of this step is to prevent phosphate in the PBSfrom causing unwanted precipitation of the uranyl acetate stain useddownstream in step (vi). Therefore, if the GV solution is veryconcentrated, direct dilution of the GV sample into the above-mentionedHEPES buffer to a final OD of 0.2 would be a quicker alternative. Next,dilute the GVs to a final OD of 0.2. Next, spin 2% Uranyl acetatesolution in a benchtop centrifuge at 14,000×g for 5 minutes to pelletany precipitate. Next, charge Formvar TEM grids using the glow dischargesystem with 15 mA current for 1 minute. Next, place 2 μl of well-mixedGV solution on the charged Formvar TEM grids for 3 minutes.

The sample should be placed on the carbon side of the grid; avoidplacing sample on the copper side. For convenience, we use PELCOreverse, anti-capillary tweezers to hold the TEM grid while addingsample and negative stain. Next, add 5 μl of 2% uranyl acetate to the GVsolution on the TEM grid for 30 seconds. Next, using a Whatman filterpaper, wick the solution by gently touching the edge of the grid. Forconsistent results, leave a thin film of sample on the grids and leaveto air dry. Finally, image the grids using TEM. Standard TEM methodsapplicable to the present disclosure are known to those skilled in theart.

In particular, negative contrast TEM can be used for imaging GV size,shape, texture and integrity following production and physical orbiochemical treatments. Negative staining with uranyl acetate can beused to produce contrast, and use of a buffer such as HEPES is preferredover phosphate buffers that may precipitate with the uranyl acetate. Theconcentration of the GV solution spotted on the grid directly correlateswith the density of GV particles on the grid.

Example 10: Exemplary GV's Engineering: Stripping of Native GvpCs fromAna Gvs

An exemplary protocol for stripping native GvpC off Ana GVs in order toprepare ΔGvpC GVs follows: First, dilute purified Ana GVs in PBS suchthat OD_(500,ps)<10. Next, prepare 3:2 (vol/vol) mix of GV strippingbuffer (10M urea in 100 mM Tris buffer) and GV solution in PBS. Pipet1.7 mL into 2 mL microcentrifuge tubes. Next, centrifuge at 300 g for 4hours, or until the subnatant is completely clear. Remove the clearsubnatant with a syringe using a 21 G flat needle. Retain the milkywhite supernatant in the tube. Resuspend the GV-containing supernatantin GV stripping Buffer (Round 2 i.e. 6M urea, 60 mM Tris-HCl). Repeatthis step 1 time. Next, confirm GvpC removal by performing SDS-PAGE.Incubate a 1:1 (vol/vol) mix of GVs in 2× Laemmli buffer (containing 5%(vol/vol) 2-mercaptoethanol) at 95° C. for 5 m. Centrifuge briefly tocollect condensate.

Next, assemble the electrophoresis cell with the comb and tape removedfrom the polyacrylamide gels. Fill the inner chamber completely with 200mL 1×TGS buffer. Ensure that the cell is not leaking fluid. Fill theouter chamber up to mark with 600 mL 1×TGS buffer. Next, load theprotein ladder and samples in the gel using gel-loading tips. GVs shouldbe at OD₅₀₀>3 prior to the 1:1 dilution. If purified proteins are beingrun on the same gel for comparison, load >100 ng. In order to preventcontamination between wells, do not exceed the maximum recommendedvolume per well. Next, connect electrophoresis cell to power supply andrun the gel for 55 m at 120V. Next, recover the gel by disassembling theelectrophoresis cell and the gel cassette. Incubate the gel in a holderwith DI H₂O for 10 m, then stain for 1 h with 10 mL SimplyBlue™SafeStain. De-stain the gel for at least 1 h with 10 mL DI H₂O. Next,image the gel using a Coomassie imaging protocol using the gel imagingsystem to visualize protein bands. The GvpC band at approximately 25 kDashould be missing.

ΔGvpC GVs can be stored in urea buffer at 4° C. for no more than 1 week.When preparing ΔGvpC GVs for long term storage without any furthergenetic functionalization or recombinant GvpC addition it can be usefulto dialyze the GV solution against PBS in order to completely remove theurea.

Example 11: Exemplary GV's Engineering: Chemical Functionalization ofAna GVs

An exemplary protocol for chemical functionalization of GVs follows: Aswould be understood by those skilled in the art, purified Ana, Halo andMega GVs contain lysine residues on the surface that can be used tochemically conjugate a variety of moieties such as polyethylene glycol,fluorophores and biotin using an amine-reactive coupling group such asN-hydroxysuccinimide ester. First, measure the concentration of purifiedGVs using the OD relationships in Table 2. Next, aliquot the NHS-moietyin anhydrous DMSO at 100× of the required molar concentration for theamine-NHS reaction. For Alexa-488-NHS conjugation to Ana GVs, aliquot 5μl of the 10 mM stock solution of the dye pre-prepared inanhydrous-DMSO. Ensure that the NHS-moiety solution does not containdetergents or surfactants that can affect the integrity and propertiesof GVs. Next, adjust the concentration and volume of GVs to the desiredamount and ensure that the buffer is free of amines (avoid Tris buffer).

For Alexa-488-NHS conjugation to Ana GVs, bring Ana GVs to OD1 in 1 mLof PBS at pH 7.4. If GVs were previously in a buffer containing freeamines or PBS with pH less than 7, ensure complete buffer exchange withPBS at pH 7-9 before proceeding with the amine-NHS reaction. Next, add10⁵ molar excess of the NHS-moiety to GVs, keeping the DMSOconcentration at 0.5% or less of the total reaction volume. ForAlexa-488-NHS conjugation to Ana GVs, add 5 μl of the 10 mMAlexa-488-NHS in DMSO to 1 mL of Ana GV solution.

Based on the average number of gvpA and gvpC protein monomers that makeup Ana GVs, approximately 50,000 lysine residues are present for everyAna GV. One can tune the molar ratio of the two reactants(NHS-moiety:GV) to achieve the desired reaction efficiency. Next, allowthe reaction to proceed for 4 hours at room temperature under gentlerotation. Next, quench the unreacted NHS-moieties using Tris-HCl bufferat pH 8 to a final concentration of 10 mM for 20 minutes at roomtemperature under gentle rotation. Next, add the whole reaction todialysis tubes (6-8 kDa cutoff) and dialyze against a 4000× volumeexcess of PBS at 4° C. for 8 hours. Replace the buffer and allowdialysis to continue for an additional 8 hours. If NHS-moiety is notamenable to dialysis, repeated rounds of centrifugally-assistedpurification is an alternative method to remove excess reactants and/orfor buffer exchange. Chemically functionalized GVs can be stored in PBSbuffer for up to one year at 4° C.

Example 12: Use of GVs as Contrast Agents for Non-Invasive ImagingApplications

GVs can be used as a contrast agent in ultrasound imaging. Theultrasound pulses can be adjusted to have a peak positive pressure basedon the acoustic collapse pressures of the GVs contained in the contrastagent.

A contrast agent comprising a plurality of GVs can be administered to atarget site, each having a distinct ultrasound collapse pressure. Thosecontrast agents can then be selectively and individually removed fromthe imaging process to produce different images that, then, can be usedfor differential contrasting (comparing/subtracting one image fromanother to highlight the differences between the two images) to providehigher structure contrast in a final image.

The acoustic collapse profiles under ultrasound are evaluated for eachGV type. GVs can be imaged in multi-well agarose phantoms at a chosenultrasound frequency while being subjected to ultrasound pulses withincreasing peak positive pressure amplitudes ranging from a lower valueto a higher value. The acoustic collapse curves can be normalized byfitting the acoustic collapse curve with a Boltzman sigmoid function.From the normalized acoustic collapse curve, a number of acousticcollapse pressure can be derived, including an initial collapsepressure, midpoint collapse pressure and complete collapse pressure. Thepeak positive pressure of the ultrasound pulses can then be selectedaccording to those derived acoustic collapse pressures.

To create a background contrast, a base image can be obtained byapplying the ultrasound pulses having a peak positive pressure lowerthan the lowest initial collapse pressure of all the GV types in thecontrast agent.

When two or more GV types are contained in the contrast agent, amaximally informative collapse pressure can be calculated based on theacoustic collapse pressure curves of a pair of spectrally adjacent GVsas described in the detailed description. The ultrasound pulses can thenbe selected to have a peak positive pressure equal to the maximallyinformative collapse pressure in order to maximally collapse one of thetwo GV types, but minimally collapse the other GV type.

An exemplary method to provide ultrasound imaging with the contrast ofthe GVs herein described is provided in FIG. 27.

Example 13: gvpC from Halobacterium salinarum, Anabaena Flos-Aquae,Halobacterium Mediterranei, Microchaete Diplosiphon and Nostoc sp

GvpC protein can be isolated GVs from Halobacterium salinarum, Anabaenaflos-aquae, Halobacterium mediterranei, Microchaete diplosiphon andNostoc sp., and others identifiable by a skilled person. DNA sequencesof gvpC proteins encoded by these species are shown in Table 3.

TABLE 3 DNA sequences of gvpC protein from exemplary species: SEQGenBank ID Species accession No. DNA Sequence NO: Anabaenaflos- X07544.1ATGATTTCTTTAATGGCAAAAATCCGGCAAGAAC 1 aquaeATCAGTCAATAGCAGAGAAAGTGGCTGAACTAT CTCTTGAGACCAGAGAATTCTTGTCCGTCACGACAGCGAAAAGACAAGAGCAAGCTGAAAAACAAG CTCAAGAACTGCAAGCATTCTACAAGGATCTTCAGGAAACAAGTCAGCAGTTTTTATCAGAAACAGC CCAAGCCAGAATTGCTCAAGCTGAAAAACAAGCTCAAGAACTGTTAGCATTCCACAAAGAACTTCAA GAAACAAGTCAGCAGTTTTTATCAGCAACAGCCCAAGCCAGAATTGCTCAAGCTGAAAAACAAGCGC AAGAACTGTTAGCATTTTATCAAGAAGTTCGGGAAACAAGTCAGCAGTTTTTATCAGCAACAGCCCAA GCAAGAATTGCTCAAGCTGAAAAACAAGCTCAAGAACTGTTAGCATTCCACAAAGAACTTCAAGAA ACAAGTCAGCAGTTTTTATCAGCAACAGCCGACGCAAGAACTGCTCAAGCTAAGGAACAGAAGGAAT CTCTCCTGAAATTCCGTCAGGATTTGTTTGTGAGTATCTTTGGTTAATAA Halo- NC_002608.1 ATGAGTGTCACAGACAAACGCGACGAGATGAGT 3bacterium GeneID: ACTGCCCGCGATAAGTTCGCAGAATCACAGCAG salinarum 1449258GAGTTCGAATCATACGCTGACGAGTTTGCAGCCG ATATCACGGCAAAGCAAGACGATGTCAGCGACCTTGTCGATGCGATCACCGACTTCCAGGCGGAGAT GACCAACACGACGGATGCATTTCACACATATGGTGACGAGTTCGCCGCTGAGGTTGACCACCTCCGTG CCGATATTGACGCCCAGCGGGACGTGATCCGTGAGATGCAGGATGCGTTCGAGGCATATGCTGACA TCTTCGCTACAGATATCGCAGACAAACAAGATATCGGCAATCTTCTGGCTGCGATTGAGGCGCTCCGA ACAGAGATGAACTCAACCCACGGGGCATTCGAAGCATATGCGGACGACTTCGCAGCCGATGTCGCTG CGCTCCGTGATATATCTGATCTGGTTGCAGCAATCGACGACTTCCAAGAGGAATTCATCGCCGTGCA GGACGCATTTGACAACTACGCTGGTGACTTCGATGCGGAGATCGACCAGCTCCACGCTGCCATCGCTG ACCAGCACGACAGCTTCGACGCTACCGCGGACGCCTTCGCAGAGTACCGAGATGAGTTCTATCGCAT AGAGGTGGAAGCACTGCTTGAGGCGATCAACGACTTCCAGCAGGACATCGGTGACTTCCGAGCGGA GTTTGAAACGACTGAGGACGCGTTCGTTGCCTTCGCCCGTGACTTCTATGGCCACGAGATCACGGCCG AGGAAGGCGCCGCCGAAGCGGAAGCCGAACCCGTCGAGGCTGACGCGGACGTCGAAGCGGAAGCAG AAGTCTCTCCAGACGAAGCTGGCGGAGAATCCGCCGGTACCGAGGAAGAAGAGACAGAGCCGGCCG AGGTGGAAACAGCGGCTCCAGAAGTAGAGGGGAGTCCTGCGGACACGGCAGACGAAGCGGAAGATA CGGAAGCAGAGGAGGAGACAGAGGAAGAGGCACCGGAAGACATGGTGCAGTGCCGGGTGTGCGGC GAATACTATCAGGCCATCACGGAGCCCCATCTCCAGACCCATGATATGACGATTCAGGAGTACCGCG ACGAGTACGGTGAGGATGTCCCCCTTCGGCCGGATGATAAAACATGA Halo- CP007551.1 ATGAGTGTCAAAGACAAACGTGAAAAGATGACC 5bacterium Protein ID: GCCACCCGCGAGGAATTCGCGGAAGTACAGCAA mediterraneiAHZ21249.1 GCGTTCGCGGCCTATGCCGACGAGTTCGCTGCCGATGTTGACGATAAACGAGACGTAAGCGAACTCG TCGATGGGATTGATACCCTGCGGACGGAGATGAACAGCACTAACGATGCGTTTCGTGCATACAGTGA GGAATTCGCCGCCGACGTCGAGCACTTCCATACGTCGGTTGCTGACCGACGCGACGCCTTCGACGCGT ATGCCGACATTTTCGCGACAGATGTCGCGGAGATGCAGGATGTGAGTGACCTCCTCGCCGCAATAGA CGACCTCCGGGCGGAGATGGACGAAACTCACGAAGCGTTCGACGCCTACGCGGATGCATTCGTGACC GACGTGGCTACCCTTCGCGATGTGTCGGACCTGCTGACGGCGATTTCGGAACTCCAGTCGGAATTCGT CTCTGTGCAGGGCGAATTTAACGGCTACGCTAGTGAGTTCGGTGCCGACATCGACCAGTTCCACGCCG TTGTCGCCGAAAAACGCGATGGTCACAAAGACGTTGCTGACGCCTTCCTCCAGTACCGAGAGGAATT TCACGGCGTCGAGGTACAGTCCTTATTGGACAACATCGCTGCCTTCCAGCGAGAAATGGGGGACTAC CGGAAAGCGTTCGAAACGACTGAGGAAGCGTTCGCCTCCTTCGCTCGCGACTTCTACGGGCAGGGCG CTGCTCCCATGGCGACACCCTTGAACAACGCGGCTGAAACAGCCGTGACTGGCACGGAGACCGAGGT AGACATACCTCCGATAGAAGACTCCGTAGAACCCGACGGTGAAGACGAGGACTCGAAAGCAGATGA CGTCGAAGCCGAAGCCGAAGTCGAGACGGTAGAAATGGAGTTCGGTGCGGAGATGGACACAGAAGC CGACGAAGACGTCCAATCGGAGTCGGTCAGAGAAGACGACCAGTTCCTGGACGACGAGACGCCAGA GGATATGGTCCAGTGTCTGGTGTGCGGCGAATATTATCAGGCGATTACAGAACCCCACCTTCAGACAC ACGACATGACGATCAAGAAATACCGCGAAGAGTACGGCGAGGACGTGCCACTCCGCCCGGATGATA AAGCATGA Microchaete X06085.1ATGACTCCTTTAATGATCAGAATCCGGCAAGAGC 7 diplosiphonATCGAGGAATAGCAGAGGAAGTAACTCAACTAT TTAAAGATACTCAAGAATTCTTGTCCGTGACCACAGCGCAAAGACAAGCGCAAGCTAAAGAACAAGC TGAAAATCTGCATCAGTTCCATAAGGATCTGGAGAAAGACACTGAAGAGTTTTTAACAGATACAGCT AAAGAAAGAATGGCTAAAGCCAAACAGCAAGCTGAAGATCTGTTCCAATTCCATAAGGAAATGGCA GAAAACACCCAAGAGTTTTTGTCAGAAACAGCTAAAGAAAGAATGGCGCAAGCTCAAGAGCAAGCT CGACAATTGCGCGAATTCCATCAAAACCTTGAGCAAACAACCAACGAATTTTTAGCTGACACAGCTA AAGAAAGAATGGCGCAAGCTCAAGAACAAAAACAACAGCTACATCAATTCCGTCAGGATTTGTTTGC TAGCATTTTTGGTACATTTTAG Nostoc sp.BA000019.2 ATGACGGCTTTAATGGTAAGAATCCGGCAAGAG 9 Protein IDCATCGGTCGATAGCTGAGGAAGTAACTCAACTAT BAB73951.1TTAGAGAGACTCATGAATTCTTGTCCGCTACAAC AGCACACAGACAAGAGCAAGCCAAACAGCAAGCGCAACAGCTACATCAGTTCCACCAAAATCTGGA GCAGACAACCCACGAGTTTTTAACAGAAACCACAACACAAAGGGTTGCTCAAGCTGAAGCACAGGC AAATTTTTTGCATAAGTTTCACCAAAATCTAGAACAGACCACCCAAGAGTTTCTAGCAGAAACAGCA AAAAACAGAACTGAGCAAGCCAAAGCACAAAGTCAATATCTGCAACAATTTCGTAAGGATTTGTTTG CTAGTATTTTTGGCACATTTTAG

The protein sequence of gvpC for these species can be found at Uniprotand other databases identifiable by skilled persons. Amino acidsequences of gvpC proteins encoded by Halobacterium salinarum, Anabaenafloc-aquae, Halobacterium mediterranei, Microchaete diplosiphon andNostoc sp are shown in Table 4.

TABLE 4 Protein sequences of gvpC from exemplary species: SEQ UniProt IDSpecies ID No. Amino acid Sequence NO: Anabaenaflos- P09413MISLMAKIRQEHQSIAEKVAELSLETREFLSVTTA 2 aquaeKRQEQAEKQAQELQAFYKDLQETSQQFLSETAQ ARIAQAEKQAQELLAFHKELQETSQQFLSATAQARIAQAEKQAQELLAFYQEVRETSQQFLSATAQAR IAQAEKQAQELLAFHKELQETSQQFLSATADARTAQAKEQKESLLKFRQDLFVSIFG Halo- P24574 MSVTDKRDEMSTARDKFAESQQEFESYADEFAAbacterium DITAKQDDVSDLVDAITDFQAEMTNTTDAFHTY  4 salinarumGDEFAAEVDHLRADIDAQRDVIREMQDAFEAYA DIFATDIADKQDIGNLLAAIEALRTEMNSTHGAFEAYADDFAADVAALRDISDLVAAIDDFQEEFIAVQ DAFDNYAGDFDAEIDQLHAAIADQHDSFDATADAFAEYRDEFYRIEVEALLEAINDFQQDIGDFRAEF ETTEDAFVAFARDFYGHEITAEEGAAEAEAEPVEADADVEAEAEVSPDEAGGESAGTEEEETEPAEVE TAAPEVEGSPADTADEAEDTEAEEETEEEAPEDMVQCRVCGEYYQAITEPHLQTHDMTIQEYRDEYG EDVPLRPDDKT Halo- Q02228MSVKDKREKMTATREEFAEVQQAFAAYADEFA  6 bacteriumADVDDKRDVSELVDGIDTLRTEMNSTNDAFRAY mediterraneiSEEFAADVEHFHTSVADRRDAFDAYADIFATDV AEMQDVSDLLAAIDDLRAEMDETHEAFDAYADAFVTDVATLRDVSDLLTAISELQSEFVSVQGEFN GYASEFGADIDQFHAVVAEKRDGHKDVADAFLQYREEFHGVEVQSLLDNIAAFQREMGDYRKAFE TTEEAFASFARDFYGQGAAPMATPLNNAAETAVTGTETEVDIPPIEDSVEPDGEDEDSKADDVEAEAE VETVEMEFGAEMDTEADEDVQSESVREDDQFLDDETPEDMVQCLVCGEYYQAITEPHLQTHDMTIK KYREEYGEDVPLRPDDKA Microchaete P08041MTPLMIRIRQEHRGIAEEVTQLFKDTQEFLSVTTA  8 diplosiphonQRQAQAKEQAENLHQFHKDLEKDTEEFLTDTAK ERMAKAKQQAEDLFQFHKEMAENTQEFLSETAKERMAQAQEQARQLREFHQNLEQTTNEFLADTAK ERMAQAQEQKQQLHQFRQDLFASIFGTFNostoc sp. Q8YUS9 MTALMVRIRQEHRSIAEEVTQLFRETHEFLSATT 10AHRQEQAKQQAQQLHQFHQNLEQTTHEFLTETT TQRVAQAEAQANFLHKFHQNLEQTTQEFLAETAKNRTEQAKAQSQYLQQFRKDLFASIFGTF

Example 14: Exemplary Methods for Producing gvpC Variant Proteins

Polynucleotides encoding gvpC DNA sequences such as those shown in Table3 can be cloned into suitable expression plasmids, such as pET28a(+)plasmid (Novagen, Temecula, Calif.) downstream of a suitable promoter,such as a T7 promoter. Additionally, one or more tags as describedherein can be encoded in the polynucleotide sequence of the plasmid toproduce a tagged gvpC protein.

Recombinant gvpC constructs such as those described herein, comprisingdeletions of nucleotides encoding one or more amino acids of the WT gvpCsequence can be made using techniques such as restriction cloning, KLDmutagenesis, or Gibson assembly, among other techniques known in theart, using commercially available reagents and lab supplies, known tothose skilled in the art.

Example 15: Expression and Purification of Ana GvpC Variants

The Ana GvpC gene sequence codon-optimized for Escherichia coliexpression was synthesized by Life Technologies, Santa Clara, Calif. AnaGvpC was cloned into a pET28a(+) plasmid (Novagen, Temecula, Calif.)downstream of a T7 promoter with an N or C-terminal His-tag. Allconstructs were made via restriction cloning, KLD mutagenesis, or Gibsonassembly using enzymes from New England Biolabs, Ipswich, Mass. Purifiedplasmids with the genetically engineered GvpC constructs weretransformed into BL21(DE3) cells (Invitrogen, Carlsbad, Calif.). Startercultures were diluted 1:250 in Terrific Broth (Sigma, St. Louis, Mo.)and allowed to reach OD₆₀₀˜0.4-0.7. Protein expression was induced byaddition of IPTG (to a final concentration of 1 mM), and cells wereharvested by centrifugation after overnight expression at 30° C.

GvpC in the form of inclusion bodies were purified by lysing the cellsusing Solulyse supplemented with DNAseI (10 μg/mL) and lysozyme (400μg/mL) at room temperature. Inclusion bodies were recovered bycentrifugation at 27,000 g for 15 min in an ultracentrifuge. Theinclusion body pellets were resuspended in 10 mM Tris-HCl buffer with500 mM NaCl and 6 M urea (pH: 8.0) and incubated with Ni-NTA resin(Qiagen, Valencia, Calif.) for 2 h at 4° C. After washing, proteins wereeluted using 250 mM imidazole. Bradford assay was used to measure theconcentration of the purified protein. Purified GvpC variants wereverified to be >95% pure by SDS-PAGE analysis.

Example 16: Ana GV Stripping and Re-Addition of Engineered GvpC Variants

Native Ana GVs were stripped of their outer GvpC layer by treatment with6 M urea solution buffered with 100 mM Tris-HCl (pH 8.5). Two rounds ofcentrifugally assisted floatation followed by removal of the subnatantlayer were done to ensure complete removal of native GvpC, as confirmedby SDS-PAGE. Stripped Ana GVs were then combined with 2× molar excess ofthe engineered GvpC variant in 6M urea buffer after accounting for a1:25 binding ratio of GvpC:GvpA. Estimating 12,768 GvpA molecules perAna GV and 564.2 pM of GVs per O.D_(PS,500) (1 cm pathlength), the molarconcentration of GvpA per O.D_(PS,500) of Ana GVs was determined to be7.2 μM and used for calculating the amount of engineered GvpC to beadded. The engineered GvpC was then allowed to slowly refold onto thesurface of the stripped Ana GVs by dialysis against 1×PBS for >12 h at4° C. using a regenerated cellulose membrane with a 6-8 kDa M.W. cutoff(Spectrum Labs, Rancho Dominguez, Calif.). Dialyzed samples weresubjected to at least 2 rounds of centrifugally assisted floatation toremove any excess unbound GvpC.

Example 17: Preparation of Recombinant GvpC Variants Engineered toAttach a HIS Tag

An exemplary protocol for preparation and purification of recombinantGvpC with a C-terminal hexahistidine tag follows: First, transform >1 ngpure plasmid encoding recombinant GvpC with a C-terminal hexahistidinetag into BL21 (DE3) competent cells and grow culture in terrific brothwith 50 μg/ml kanamycin overnight. Next, dilute 500 μL of the starterculture 1:1 with 50% glycerol in water and store at −80° C. Futurestarter cultures can be grown from aliquots of this glycerol stockinstead of fresh transformations. Next, dilute starter culture 1:250 interrific broth with 50 μg/ml kanamycin and grow to OD₆₀₀˜0.4-0.7 withshaking (250 rpm) at 37° C. Induce at a final concentration of 1 mMIPTG. Grow culture for 6-12 h at 30° C. with shaking. Next, pellet thecells in ultracentrifuge tubes at 5,500 g for 15 min at 4° C. anddiscard the supernatant. Cell pellets can be stored at −20° C. Proteinextraction is typically more effective with frozen cells. Next,resuspend the pellets in 10 mL Solulyse with 10 μg/mL DNAse. Rotate atroom temperature for 10 min. Next, centrifuge at 20,000 g for 15 m at 4°C. to clear the lysate and discard the supernatant. Resuspend the pelletin 10 mL Solulyse and lysozyme (0.25 mg/mL). Rotate at room temperaturefor 10 m. Add 5 mL Solulyse and vortex. Centrifuge at 20,000 g for 20 mat 4° C. and discard the supernatant. Thoroughly resuspend the pellet in5 mL of inclusion body solubilization buffer. Centrifuge at 20,000 g for20 m at 4° C. Add 1.5 mL Ni-NTA slurry to the supernatant, incubate at4° C. with shaking (60 rpm) for 2 h or more. Pour into a polyprep columnand collect all the flow-through, wash and elutions in the next steps.Collecting all fractions is good practice for troubleshooting andanalyzing purification steps using SDS-PAGE. Wash with 10 column volumesof inclusion body wash buffer. Elute with 2 column volumes of inclusionbody elution buffer. To quantify the eluted protein using the Bradfordassay, prepare a standard curve of bovine serum albumin (BSA) at a finalconcentrations of 100, 250, 500, 750, 1000 and 1500 μg/mL in 60 μL of 3×diluted inclusion body elution buffer in PBS. Prepare dilutions ofeluted protein 1:2 in PBS with a final volume 60 μL. Prepare a negativecontrol of 3× diluted inclusion body elution buffer in PBS. To 25 μL ofthe sample and BSA standards, add 1 mL of Bradford reagent, vortex andincubate at room temperature for 5-10 m. Prepare all samples induplicate. Blank the spectrophotometer with a negative control sampleand measure the OD₅₉₅. Measure the OD₅₉₅ of the standard curve samples.Plot the OD₅₉₅ versus the concentration, and compute linear regressionfit. Measure the OD₅₉₅ of the eluted protein samples. Use the linear fitfrom Step 37 to compute the unknown concentrations, and multiply by 3(dilution factor) to obtain concentration of stock elution solution. Theelutions can be stored separately at 4° C. Elution 1 has >80% ofcollected pure protein and is used for the subsequent experiments.Elution 2 is more dilute and is typically stored as backup or forrunning protein controls for SDS-PAGE.

Example 18: Preparation of Recombinant GvpC Variants Engineered toAttach a Spy Catch Tag

An exemplary protocol for preparation of SpyCatcher-functionalized GVsfollows: Mix SpyTag-functionalized GVs with SpyCatcher-fused proteinsaccording to the formulation: 2*OD*395 nM*volume (in L) of SpyTagGVs=nmol SpyCatcher-fused protein. This results in a 2-fold excess ofSpyCatcher to SpyTag in the reaction, based on the stoichiometrydescribed in Step 39. Note that the SpyCatcher-mNeonGreen (SC-mNG)fusion protein used in [1] is expressed separately in E. Coli asdescribed herein and using a suitable the plasmid containing SC-mNG(details in the reagents section). SC-mNG is expressed as a solubleprotein and hexahistidine-tagged, enabling purification using the sameNi-NTA slurry used for recombinant GvpC purification. Unlike GvpCinclusion bodies, soluble proteins are in the supernatant after celllysis with Solulyse/DNAse (Step 24), allowing direct incubation of thesupernatant with the slurry (Step 30). Wash and elution (Steps 31-33)are performed with soluble protein wash buffer and soluble proteinelution buffer respectively, and the protein is desalted into PBS usingPD10 desalting columns. Protein quantification is done using the Pierceor Bradford assay before use. Incubate 1 hour or more at roomtemperature. Centrifuge at 300 g for 3 hours or until subnatant isclear. Remove clear subnatant with syringe with 21.5 G needle. Retainmilky white supernatant in the tube. Resuspend supernatant in PBS.Repeat this centrifugation step one time. Store at 4° C.

Example 19: Preparation of Recombinant GvpC Variants

GVs comprising recombinant gvpC can be prepared using any method knownin the art. An exemplary protocol for preparation of GVs withrecombinant GvpC follows: Add recombinant GvpC to ΔGvpC GVs according tothe formulation: 2*OD*198 nM*volume (in L) of GVs=nmol recombinant GvpC.This provides a 2-fold stoichiometric excess of GvpC relative to bindingsites on an average Ana GV, assuming a 1:25 molar ratio of GvpC:GvpAbinding based on previous work[27]. The exact volume of recombinant GvpCto be added is calculated based on the molar mass of the particularvariant and the concentration of eluted GvpC (measured by Bradfordassay). For truncated GvpC variants with a lower GV binding affinity, ahigher stoichiometric excess may be added to promote attachment of GvpCto the GV surface. However, note that adding too much excess of GvpCmight lead to protein aggregation during dialysis. Soak the dialysistubing in PBS for 5 minutes. Add samples (GVs+recombinant GvpC) intodialysis tubing and clip both sides. Dialyze in 4 L PBS with stirring onlow speed for at least 12 h. The length of dialysis tubing used for eachsample depends on the total volume of the dialysate, which is determinedby the amount of engineered GVs required for the end application. Thetype of dialysis tubing used (molecular weight cutoff) can changedepending on the GvpC variant, as truncated variants may have a muchlower molecular weight. Transfer the dialysate into 2-mL centrifugetubes and spin at 300 g for 3 hours, or until subnatant is clear. Removesubnatant with syringe with a 21.5 G flat needle. Retain the milky whitesupernatant in the tube. Resuspend GVs in PBS. Repeat thiscentrifugation step one time. The GVs can be stored at 4° C.

Example 20: Modular Genetic Engineering of Acoustic Protein Structures

To enable modular molecular engineering of Ana GVs, a platform wasestablished in which genetically engineered GvpC variants arerecombinantly expressed in Escherichia coli and subsequently added toAna GVs that have been purified from Anabaena floc-aquae and stripped oftheir native GvpC proteins (FIG. 2 Panel d). The GVs were isolated byhypertonic and detergent-mediated lysis, followed by purification withcentrifugally assisted floatation. Native GvpC was removed by treatingthe GVs with 6M urea, which leaves the GvpA-based shell intact.[28, 29]Genetically engineered variants of Ana GvpC were produced containing N-or C-terminal hexahistidine sequences in Escherichia coli and purifiedthe resulting inclusion bodies by nickel chromatography in 6M urea.Dialysis of recombinant GvpC in the presence of stripped Ana GVs intophysiological buffer resulted in Ana GVs with a new, engineered GvpClayer (FIG. 2 Panel d). SDS-PAGE analysis confirmed the complete removalof GvpC from native Ana GVs and the re-addition of engineered proteins(FIG. 3).

Example 21: Tuning of Collapse Pressure for Acoustic Multiplexing

The gaseous interior of GVs can be collapsed with hydrostatic andacoustic pressure, erasing their ultrasound scattering signal andenabling multiplexed imaging of GVs with distinct collapse pressurethresholds.[30] To determine whether genetic tuning could enableenhanced multiplexing, three Ana GV variants were engineered withdistinct mechanical properties. ΔGvpC comprises GVs completely lackingthe outer GvpC layer; ΔN&C contains a truncated form of GvpC without itsN- and C-terminal regions; GvpC_(WT) has an engineered GvpC protein thatclosely resembles the wild-type sequence (FIG. 4 Panel a). Thehydrostatic collapse behavior of these structures was assessed usingpressurized absorbance spectroscopy, in which the optical density of GVs(which scatter 500 nm light when intact) is measured under increasinghydrostatic pressure. This provides a rapid assessment of GV mechanicsand allows comparisons to literature [3]. These three variants spanned adynamic range of 380 kPa (FIG. 4 Panel b, Table 5). ΔGvpC had the lowestcollapse pressure midpoint at 195.3±0.3 kPa, the ΔN&C variant showed anintermediate value of 374.3±1 kPa and GvpC_(WT) had the highest value of569.9±4 kPa (Table 5, N=7, ±SEM). To ensure that the decrease incollapse pressure for the ΔN&C variant was not due to unsaturatedbinding caused by reduced affinity of this GvpC variant for GvpA,collapse midpoints were measured as a function of re-added GvpCconcentration and confirmed that binding was near saturation (FIG. 5,FIG. 6).

Table 5 shows hydrostatic midpoint of collapse for engineered Ana GVsused in acoustic multiplexing experiments (FIG. 4 Panel b). The data wasfitted with a Boltzmann sigmoid function of the form ƒ(p)=(1+e^((p-p)^(c) ^()/Δp))⁻¹ with p_(c) representing the average midpoint ofcollapse. Fit parameters and R² values for each of the GV variants areprovided in the table.

TABLE 5 Hydrostatic midpoint of collapse for engineered Ana GVs used inacoustic multiplexing experiments Midpoint GV of Collapse P_(c) (SEM) ΔP(SEM) Adj. Variants (P_(c)) (kPa) (kPa) ΔP (kPa) (kPa) R-Square ΔGvpC195.30 0.27 17.01 0.24 0.999 ΔN&C 374.30 1.01 41.46 0.89 0.999 GvpC_(WT)569.85 3.64 84.87 3.21 0.992

Additional exemplary gvpC variants were engineered, the aligned proteinsequences of which are shown in FIG. 11, and as described in Table 6.

TABLE 6 Exemplary gvpC variants Ana gvpC Variant Engineered proteinfeatures ΔN&C-CERY1 GvpC comprising deletions of N- and C-terminalregions, and an ERY1 tag fused to the C- terminus of gvpC and a His-tagin the N-terminal region ΔNterm GvpC comprising a deletion of N-terminalregion and a His-tag fused to the C-terminus N-rep3-C GvpC comprisingN-terminal region, repeat 3, and C-terminal region, and a His-tag fusedto the C-terminus SR3CERY1 GvpC comprising N-terminal region containinga His-tag, repeat 3, and an ERY1 tag fused to the C-terminus WTCERY1GvpC comprising a His-tag in the N-terminal region, repeat regionsandanERY1 tag fused to the C-terminus N-rep1-C GvpC comprising N-terminalregion, repeat 1, a C-terminal region and a His-tag fused to theC-terminus SR1CERY1 GvpC comprising N-terminal region, repeat 1, andC-terminal region, a His-tag in the N- terminal region and anERY1 tagfused to the C-terminus ΔN&C GvpC comprising deletions of N- andC-terminal regions and a His-tag fused to the C- terminusN-rep2endto3mid-C GvpC comprising N-terminal region, a region from amidpoint of repeat 2 to within a midpoint of repeat 3, and C-terminalregion, and a His-tag fused to the C-terminus N-His-GvpC GvpC comprisinga His-tag in the N-terminal region ΔCterm GvpC comprising a deletion ofC-terminal region and a His-tag fused to thelast repeat GvpCWT-ACPP GvpCcomprising a His-tag in the N-terminal region and an ACPP tag fused tothe C- terminus GvpCWT-hPRM GvpC comprising a His-tag in the N-terminalregion and an hPRM tag fused to the C- terminus GvpCWT-LRP GvpCcomprising a His-tag in the N-terminal region and an LRP tag fused tothe C-terminus GvpCWT-mCD47 GvpC comprising a His-tag in the N-terminalregion and a mCD47 tag fused to the C- terminus GvpCWT-R8 GvpCcomprising a His-tag in the N-terminal region and an R8 tag fused to theC-terminus GvpCWT-RGD GvpC comprising a His-tag in the N-terminal regionand an RGD tag fused to the C- terminus GvpCWT-RDG GvpC comprising aHis-tag in the N-terminal region and an RDG tag fused to the C- terminusGvpC-SpyTag GvpC comprising a SpyTag fused to the C-terminus, and aHis-tag fused to the C-terminus of the SpyTag N-rep1to3-C GvpCcomprising an N-terminal region, repeats 1 to 3, a C-terminal region,and a His-tag fused to the C-terminus N-rep1to2-C GvpC comprising anN-terminal region, repeats 1 to 2, a C-terminal region, and a His-tagfused to the C-terminus N-rep1to4-C GvpC comprising an N-terminalregion, repeats 1 to 4, a C-terminal region, and a His-tag fused to theC-terminus GvpCWT GvpC comprising a His-tag fused to the C-terminus

FIG. 18 shows a predicted alignment of the tandem repeat regions (Rep)in gvpC from Anabaena floc-aquae, wherein the five repeat regions 1-5(rep1, rep2, rep3, rep4, and rep5) are preceded by an upstreamN-terminal region and are followed by a downstream C-terminal region.Some of the variants described here were produced by deletions ofvarious parts of the N- and/or C-terminal regions, or one or more repeatregions, as described in Table 6.

The hydrostatic collapse behavior of the additional gvpC variantsstructures was assessed, as above, using pressurized absorbancespectroscopy. FIG. 19 shows that Rep1 (also described herein asN-rep1-C) strengthens GVs more than Rep2to3 (also described herein asN-rep2endto3mid-C) or Rep3 (also described herein as N-rep3-C). FIG. 20shows that all three variants Rep1, Rep2to3 and Rep3 bind to GVs,indicating that gvpc binding to GVs is not sufficient to strengthen GVs,but binding of different gvpc variants impart different strengthening toGVs. In addition to GVs comprising the ΔN&C variant described above(also described herein as Δnose/tail), hydrostatic collapse behavior ofGVs comprising additional variants Δnose (also described herein asΔNterm) and Δtail (also described herein as ΔCterm) were assessed (FIG.21), and show that truncating the N-terminus of gvpc decreases collapsepressure to a greater extent than truncating the C-terminal tail, anddeleting N&C at the same time produces GVs with lower collapse pressurethan either truncation done separately. Hydrostatic collapse behavior ofGVs comprising variants gvpcΔ4, gvpcΔ3, gvpcΔ2, and gvpcΔ1 were alsoassessed (FIG. 21).

The data on all variants described herein are summarized in FIG. 17.FIG. 17 also shows that adding molar excess of GvpC prior to dialysisincreases collapse pressure up to a certain threshold, above which thecollapse pressure plateaus and does not increase any further (also seeFIG. 5 and FIG. 6). Furthermore, appending a His-Tag (6 amino acids) tothe N-terminus of the wild-type GvpC sequence does not cause asubstantial change in collapse pressure. Deletion of 5th and 4th repeathas little effect on collapse, while deletion of 2nd repeat has a muchbigger effect. Thus, removing repeats from the C-terminal end typicallyhas a lower effect on the collapse pressure compared to removing repeatsclose to the N-terminal end.

Overall, deletions of repeats cause less decrease of collapse pressurethan deletions to the terminal regions of GvpC. N-terminal repeatsappear to be important for GvpC-based strengthening, in particular, asdeletion of repeat 2 decreases collapse pressure by ˜150 kPa, whiledeletion of repeats 5 and 4 do not have a substantial impact on collapsepressure, and the observation that repeat 3 (consensus repeat sequence)binds to GVs but does not strengthen, while repeat 1 binds andstrengthens GVs.

Next, collapse profiles under ultrasound were evaluated under. GVs wereimaged in multi-well agarose phantoms at 6.25 MHz while being subjectedto ultrasound pulses with increasing peak positive pressure amplitudesranging from 290 kPa to 1.23 MPa.

The acoustic collapse curve can be normalized by fitting the acousticcollapse curve with a Boltzmann sigmoid function. Exemplary parametersfor fitting the acoustic collapse curve with the Boltzmann sigmoidfunction can be found in Table 7. Table 7 also lists the acousticmidpoint of collapse for engineered Ana GVs used in multiplexingexperiments (FIG. 4 Panel c).

TABLE 7 Exemplary parameters used in Boltzmann sigmoid function fittingMidpoint of Collapse P_(c) (SEM) ΔP ΔP (SEM) Adj. R- GV Variants (Pc)(kPa) (kPa) (kPa) (kPa) Square ΔGvpC 571.00 1.51 14.48 1.03 0.998 ΔN&C657.04 3.94 77.47 3.70 0.997 GvpC_(WT) 868.81 6.56 94.00 5.57 0.994

The data was fitted with the form ƒ(p)=+e^((p-p) ^(c) ^()/Δp))⁻¹ withp_(c) representing the average midpoint of collapse. Fit parameters andR² values for each of the GV variants are provided in the table.

Similar to trends observed for hydrostatic collapse, the ΔGvpC variantcollapses under the lowest acoustic pressure, followed by ΔN&C andGvpC_(WT) (FIG. 4 Panel c, Table 7). Notably, the collapse midpoints inthe acoustic regime were substantially higher than in the hydrostaticregime. This is explained by GVs having a gas efflux time ofapproximately 1.5 μs, [31] which is too slow for gas molecules containedin the GV to exit the structure during the 80 ns positive half-cycle of6.25 MHz ultrasound, allowing the gas to compressively reinforce the GVshell. On the other hand, under hydrostatic conditions, pressure changesoccur on the time scale of seconds, allowing gas molecules to exit theGV during pressurization and resulting in the shell carrying the fullcompressive load by itself. [32] It is also noted that the acousticcollapse curves appear somewhat more closely spaced than hydrostaticcollapse curves, which can be explained by the applied acoustic pressurefield having a non-uniform profile over the imaged GV sample. Fitting aBoltzmann sigmoidal function to these collapse curves reveals a uniqueacoustic collapse spectrum for each engineered GV (FIG. 4 Panel d).

To take advantage of the distinct acoustic collapse spectra of differentGV variants for multiplexed imaging, pressure spectral unmixing was usedto obtain multiplexed images of our three GV variants. FIG. 4 Panel fshows ultrasound images taken at a non-destructive baseline pressurebefore and after exposing the GV samples to three sequentiallyincreasing collapse pulses. The spectrally unmixed images (FIG. 4 Panelg) uniquely identify acoustic signals from each GV variant. FIG. 7 showsthe matrix of coefficients used to generate these images. Thiscombination of engineered GVs and pressure spectral unmixing can beuseful in many scenarios requiring ultrasound imaging of multiplemolecular targets in the same sample.

Example 22: Maximally Informative Acoustic Pressure of a GV in Mixtureof GV Variants

For any two spectrally adjacent GVs, the maximally informative pressurerefers to as an acoustic pressure that can maximize the collapse of oneGV but minimize the collapse of the other GV.

As shown in FIG. 4, panel d, ΔGvpC and ΔN&C are two spectrally adjacentGVs, ΔN&C and GvpC_(WT) are two spectrally adjacent GVs, while ΔGvpC andGvpC_(WT) are not spectrally adjacent.

The maximally informative acoustic pressure can be measured byconstructing an acoustic collapse curve for each GV used in the contrastagent as shown in FIG. 4, panel c as described above. The maximallyinformative pressure between the two GV types can be calculated based onfitted sigmoid function ƒ(p). For two GV types having a fitted sigmoidf₁(p) and f₂(p), the maximally informative collapse pressure, p_(max),is chosen so as to maximize the difference between f₁(p_(max)) andf₂(p_(max)).

For example, as shown in FIG. 4, panel d, the first maximal informativeacoustic pressure applied during the multiplexing ultrasound imaging isestimated as 630 kPa. The first maximal informative acoustic pressure iscapable of maximally collapsing the ΔGvpC variant while minimallycollapsing the other two variants, i.e. ΔN&C and GvpC_(WT). The secondmaximal informative acoustic pressure is estimated as 790 kPa. Thesecond maximal informative acoustic pressure is capable of maximallycollapsing the ΔN&C variant while minimally collapsing the remainingvariant, i.e. GvpC_(WT). The third, and the last, maximal informativeacoustic pressure is 1230 kPa. This maximal informative acousticpressure is applied to collapse the remaining GvpC_(WT) variant, i.e. itis greater than the acoustic pressures plotted on the GvpC_(WT) curve inFIG. 4, panel d.

Example 23: Modulation of Harmonic Ultrasound Signals

Non-linear signals from ultrasound contrast agents can dramaticallyenhance their ability to be distinguished from background tissues, whichmainly scatter linearly. [33, 34] In the initial description of gasvesicles as ultrasound reporters, it was found that GVs fromHalobacterium salinarum (Halo GVs) produce strong non-linear signals inthe form of harmonics of the insonation frequency, while Ana GVs show noharmonic response.[30] Since Halo GVs also have a significantly lowercritical collapse pressure than Ana GVs,[3] it is hypothesized thataltering Ana GV shell mechanics by engineering GvpC could yield Ana GVswith harmonic signals.

Accordingly, the frequency response of engineered Ana GVs to 4.46 MHzpulses over a receive bandwidth of 2-10 MHz was characterized.Consistent with the hypothesis, ΔGvpC showed a sharp peak at the secondharmonic frequency of 8.9 MHz in addition to the fundamental peak at thetransmitted frequency, while GvpC_(WT) showed only a linear signal (FIG.8 Panel a). Ultrasound images formed by bandpass filtering around thefundamental and second harmonic frequencies showed a substantialdifference in the harmonic acoustic response of GV variants (p<0.01,N=7, paired t-test), for the same level of fundamental signal (FIG. 8Panel b-e). The harmonic signals from ΔGvpC were 3.71-fold higher thanGvpC_(WT) (FIG. 8 Panel e). These results demonstrate for the first timethat protein engineering can be used to modulate the acoustic propertiesof a structure.

To show that engineered Ana GV variants are capable of producingharmonic signals in vivo, intravenous injections of the ΔGvpC andGvpC_(WT) variants into live, anaesthetized mice were performed.Ultrasound imaging of the inferior vena cava (IVC) was performed infundamental and second-harmonic modes (transmission at 4.46 MHz andreception filtered around 4.46 MHz and 8.9 MHz center frequencies,respectively). FIG. 9 Panel a provides a schematic illustration of thein vivo experiment. Five seconds after the start of the injection,enhanced non-linear signals were observed for the ΔGvpC variant comparedto GvpC_(WT), while their fundamental signals were comparable (FIG. 9,Panel b-d). Repeated trials showed a statistically significantdifference (p<0.01, N=6, paired t-test) in the harmonic response of thetwo variants for the same level of fundamental signal (FIG. 9 Panel e),consistent with in vitro results. The ability to genetically tune theharmonic properties of GV contrast agents will dramatically enhancetheir utility for in vitro and in vivo imaging.

Example 24: Tuning of Surface Charge, Targeting Specificity andMultimodal Imaging

After demonstrating the ability of GvpC to serve as a genetic platformfor tuning the mechanical and acoustic properties of GVs, its capacityto enable the engineering of GV surface properties and add functionalitywas examined.

To do so, the C-terminus of GvpC was used as modular sites for proteinfusion (FIG. 10 Panel a, FIG. 11). As a first proof of concept, theability of GvpC fusions to modulate GV surface charge was tested, animportant property that influences the behavior of structures insolution and in vivo.[35] GvpC was fused with the lysine rich protein(LRP), which contains 100 positive charges at physiological pH.Re-addition of this protein to GVs resulted in structures with 28±4 mVhigher zeta potential compared to GvpC_(WT) (FIG. 10 Panel b).

Next, the ability of GvpC fusions to endow GVs with functionality forspecific cellular targeting was tested. A well-studiedreceptor-targeting peptide is RGD, which binds effectively to a widerange of integrins.[36] GVs engineered to express GvpC_(RGD) on theirsurface were compared with wild type GvpC and scrambled GvpC_(RDG)controls in terms of their ability to target the integrin-overexpressingU87 glioblastoma cell line in vitro. The GVs were chemically conjugatedwith the Alexa Fluor-488 fluorophore for visualization using confocalmicroscopy. GVs functionalized with RGD exhibited a marked increase incell binding compared to controls (FIG. 10 Panel c, d). This techniquepresents a generalizable approach for future studies targeting GVs tomolecular markers in vivo.

Using a similar engineering strategy, GvpC fusions was created tomodulate the interaction of GVs with macrophages, which are both imagingtargets and important actors in nanoparticle clearance from circulation.CD47, present on endogenous cell membranes in humans, mice, and othermammals, is a well-studied putative marker of self Discher andcolleagues recently described a minimized peptide from the human CD47protein, dubbed the ‘self’ peptide, which led to reduced uptake of cellsand nanoparticles by the mononuclear phagocytic system.[37] On the otherhand, polycationic peptides such as polyarginine (R8) promote particleuptake by phagocytic cells.[38] By fusing each of these molecules toGvpC, we tested whether genetic engineering could modulate GV uptake inRAW 264.7 murine macrophages. As visualized by confocal microscopy, GVsgenetically functionalized with GvpC_(mcD47) showed reduced macrophageuptake compared to GVs with GvpC_(WT). On the other hand, GVsfunctionalized with GvpC_(R8) were taken up much more efficiently (FIG.10 Panel e, 1). These molecular strategies can be used in future studiesto enable cellular labeling for in vivo tracking applications or toenhance the circulation lifetime of targeted GVs.

Finally, to further simplify future GV functionalization, a highlymodular approach was developed, in which the GV surface can bespecifically covalently conjugated to other recombinant proteins througha facile process that does not involve urea treatment and dialysis. Toachieve this goal, GvpC was fused with SpyTag (ST), a 13-residue peptidethat forms a covalent amide bond with a partner SpyCatcher protein underphysiological conditions.[39] This system allows SpyTagged GVs to befunctionalized with SpyCatcher fusions in a rapid biocompatiblereaction. It was found that GvpC_(WT) binds to GVs with similarstoichiometry to GvpC_(WT) and provides reinforcement againstpressure-induced collapse (FIG. 12). Each modified GV had an average of1,000 SpyTag functionalities (FIG. 13). To demonstrate the utility ofthis modular functionalization approach, these GVs were reacted with therecombinantly expressed fluorescent protein SpyCatcher-mNeonGreen(SC-mNG) to enable multimodal acoustic and fluorescent imaging. Theresulting fluorescent GVs were purified by buoyancy enrichment. SDS-PAGEanalysis confirmed SpyTag-SpyCatcher covalent bond formation (FIG. 14),and FIG. 10 Panel g shows multimodal imaging of mNG-labeled GVs withultrasound and fluorescence. The ultrasound images show similarechogenicity between fluorescently-labeled GVs, wild-type and unreactedcontrols. GvpC_(WT) Ana GVs do not show any fluorescence after reactionwith SC-mNG (FIG. 10 Panel g), highlighting the specificity of theSpyTag-SpyCatcher reaction and confirming that buoyancy enrichmenteliminates unreacted fluorescent proteins (FIG. 14). Notably, labeledST-GVs remain fluorescent after acoustic pressure-induced collapse,which may be useful for follow-up histological examinations afterultrasound imaging. These results establish the GvpC_(ST)-SpyCatchersystem as a highly modular and convenient approach to generatefunctionalized GVs, thereby enabling the first dual-mode imaging ofthese structures.

As shown in FIG. 17, attachments to the C-terminus appear to betolerated well as it does not cause substantial decrease in collapsepressure as compared deleting the terminal regions. However, appendingfunctional tag residues to the C-terminus of GvpC_(WT) reduces collapsepressure depending on the length and exact properties of the amino acidsequence. Small sequences such as RGD and RDG do not appear to affectthe collapse pressure to a substantial extent, however mCD47 and longertags such as LRP (100 residues) reduce it to a greater extent. Also,Spytag-Spycatcher is a suitable method to functionalize GVs with bigmolecules such as fluorescent proteins without changing their collapsepressure (mechanical and acoustic properties).

Example 25: Engineering gvpC Variants in Species Other than Anabaenaflos-aquae

It is expected that gvpC in other species can be engineered to producevariants in a similar manner to that described herein for Anabaenafloc-aquae.

Evidence suggests that gvpC from different species may be functionallysimilar. For example, evidence from Buchholz et al. (1993) showed GVsfrom 3 different species can be stripped of its native GvpC to decreasehydrostatic collapse, and wild-type Anabaena GvpC can be re-added to allof them to enable an almost complete recovery of collapse pressure [27]

In addition, FIG. 16 shows the wild-type amino acid GvpC sequences from5 different organisms, where Panel a shows Halobacterium salinarum gvpC,Panel b shows Anabaena flos-aquae gvpC, Panel c shows Halobacteriummediterranei gvpC, Panel d shows Microchaete diplosiphon gvpC, and andPanel e shows Nostoc sp. gvpC. In each panel, sequences are shown withpredicted aligned of the tandem repeat regions (Rep) within each gvpCprotein, preceded by the N-terminal region (N-term) and followed by theC-terminal region (C-term).

Based on the structural similarity of gvpC from these five species,comprising repeated regions flanked by N- and C-terminal regions, it isexpected that, as in Anabaena floc-aquae gvpC, engineering gvpC fromthese other species will enable production of variants that have alteredhydrostatic and acoustic collapse pressure values and altered harmonicbehavior compared to the wild-type gvpC, that will be useful for imagingapplications. In particular, it is expected that removing repeats fromthe C-terminal end will typically have a lower effect on the collapsepressure compared to removing repeats close to the N-terminal end, thattruncating the N-terminus of gvpc from these other species will decreasecollapse pressure to a greater extent than truncating the C-terminaltail, and deleting N&C at the same time will produce GVs with lowercollapse pressure than either truncation done separately.

It is also expected that adding molar excess of GvpC compared to gvpAprior to dialysis increases collapse pressure up to a certain threshold,above which the collapse pressure plateaus and does not increase anyfurther. In addition, it is expected that appending a His-Tag to theN-terminus of the wild-type GvpC from these other species does not causea substantial change in collapse pressure.

Appending tags to gvpC from these other species is also expected toresult in functionalized gvpC variants with similar behaviors asdescribed herein for gvpC from Anabaena floc-aquae.

Example 26: Exemplary GVs Producing Microorganisms

FIG. 22 shows a list of GV producing microbes and FIG. 23 describes thedifferent genes present in the gene cluster for haloarchael GVs (whichhave the largest number of different gyp genes) and their predictedfunction and features.

Example 27: Correspondence Between Hydrostatic Collapse Pressure andAcoustic Collapse Pressure

In this example, hydrostatic and acoustic collapse pressures weremeasured for four GV types: Ana ΔGvpC, Ana ΔN&C, Ana WT, and Halo WT.The data is shown in Table 8.

TABLE 8 Measurement of hydrostatic and acoustic collapse pressures forfour GV types Hydrostatic Acoustic Collapse GV Type Collapse MidpointMidpoint Ana ΔGvpC 195.3 571 Ana ΔN&C 374.3 657.04 Ana WT 569.85 868.81Halo 59 550

The collapse pressure correspondence between acoustic collapse pressuremidpoints and hydrostatic collapse pressure midpoints for Ana GVs isplotted in FIG. 24, in which C is 395 and M is 0.8.

The collapse pressure correspondence between acoustic collapse pressuremidpoints and hydrostatic collapse pressure midpoints for Halo GVs isplotted in FIG. 25. C is 475 and M is 0.64.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the materials, compositions, systems andmethods of the disclosure, and are not intended to limit the scope ofwhat the inventors regard as their disclosure. Those skilled in the artwill recognize how to adapt the features of the exemplified methods andarrangements to additional gas vesicles, related components, genetic orchemical variants, as well as in compositions, methods and systemsherein described, in according to various embodiments and scope of theclaims.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe disclosure pertains.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background, Summary, Detailed Description, andExamples is hereby incorporated herein by reference. All referencescited in this disclosure are incorporated by reference to the sameextent as if each reference had been incorporated by reference in itsentirety individually. However, if any inconsistency arises between acited reference and the present disclosure, the present disclosure takesprecedence. Further, the computer readable form of the sequence listingof the ASCII text file P2049-US-Seq-List-ST25 is incorporated herein byreference in its entirety.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe disclosure claimed. Thus, it should be understood that although thedisclosure has been specifically disclosed by embodiments, exemplaryembodiments and optional features, modification and variation of theconcepts herein disclosed can be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this disclosure as defined by the appended claims.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessthe content clearly dictates otherwise. The term “plurality” includestwo or more referents unless the content clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and possible subcombinationsof the group are intended to be individually included in the disclosure.Every combination of components or materials described or exemplifiedherein can be used to practice the disclosure, unless otherwise stated.One of ordinary skill in the art will appreciate that methods, deviceelements, and materials other than those specifically exemplified may beemployed in the practice of the disclosure without resort to undueexperimentation. All art-known functional equivalents, of any suchmethods, device elements, and materials are intended to be included inthis disclosure. Whenever a range is given in the specification, forexample, a temperature range, a frequency range, a time range, or acomposition range, all intermediate ranges and all subranges, as wellas, all individual values included in the ranges given are intended tobe included in the disclosure. Any one or more individual members of arange or group disclosed herein may be excluded from a claim of thisdisclosure. The disclosure illustratively described herein suitably maybe practiced in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

A number of embodiments of the disclosure have been described. Thespecific embodiments provided herein are examples of useful embodimentsof the invention and it will be apparent to one skilled in the art thatthe disclosure can be carried out using a large number of variations ofthe devices, device components, methods steps set forth in the presentdescription. As will be obvious to one of skill in the art, methods anddevices useful for the present methods may include a large number ofoptional composition and processing elements and steps.

In particular, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

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The invention claimed is:
 1. An ultrasound imaging method to be used ona target site contrasted with a gas vesicle protein structure (GVPS)type having a selectable acoustic collapse pressure value derived froman acoustic collapse pressure profile of the GVPS type and a hydrostaticcollapse pressure profile, a midpoint of the acoustic collapse pressureprofile higher than a midpoint of the hydrostatic collapse pressureprofile, the method comprising collapsing the GVPS type by applyingcollapsing ultrasound to the target site, the collapsing ultrasoundapplied at a collapsing ultrasound pressure greater than the selectableacoustic collapse pressure value; and imaging the target site byapplying imaging ultrasound to the target site, the imaging ultrasoundapplied at a first imaging ultrasound pressure selected to provide anuncontrasted image of the target site.
 2. The ultrasound imaging methodof claim 1, wherein the collapsing ultrasound pressure is higher thanthe midpoint of the hydrostatic collapse pressure profile.
 3. Theultrasound imaging method of claim 1, wherein the collapsing ultrasoundpressure is higher than the midpoint of the acoustic collapse pressureprofile.
 4. The ultrasound imaging method of claim 1, further comprisingfurther imaging the target site prior to the collapsing by applyingimaging ultrasound to the target site, the imaging ultrasound applied ata second imaging ultrasound pressure lower than the selectable acousticcollapse pressure and selected to provide a visible image; and comparingthe visible image of the target site with the uncontrasted image.
 5. Theultrasound imaging method of claim 4, wherein the first imagingultrasound pressure is equal to the second imaging ultrasound pressure.6. The ultrasound imaging method of claim 1, wherein the GVPS is a GVPSfrom a species of Anabaena bacteria or Halobacterium archaea.
 7. Theultrasound imaging method of claim 6, wherein the GVPS is a GVPS fromAnabaena flos-aquae, Halobacterium salinarum or Bacillus megaterium. 8.The ultrasound imaging method of claim 1, wherein the GVPS comprise aGvpC protein selected from the group consisting of ΔGvpC, ΔN&C, andGvpC_(WT), and the selectable acoustic collapse pressure value is 571kPa for ΔGvpC, 657 kPa for ΔN&C, and 869 kPa for GvpC_(WT).
 9. Theultrasound imaging method of claim 1, further comprising an initialpreparation step of administering to the target site a contrast agentcomprising the gas vesicle protein structure.
 10. The ultrasound imagingmethod of claim 1, wherein the GVPS type comprises a first gas vesicleprotein structure (GVPS) type exhibiting a first acoustic collapsepressure profile and a first selectable acoustic collapse pressure valueand a second GVPS type exhibiting a second acoustic collapse pressureprofile and-a second selectable acoustic collapse pressure value, eachGVPS type exhibiting a different acoustic collapse pressure profiledefined as a collapse function from which a collapse amount can bedetermined and a different selectable acoustic collapse pressure valuefrom their corresponding acoustic collapse pressure profile, wherein thecollapsing is performed by selectively collapsing the first GVPS type byapplying collapsing ultrasound to the target site, the collapsingultrasound applied at a first acoustic collapse pressure value equal toor higher than the first selectable acoustic collapse pressure value andlower than the second selectable acoustic collapse pressure value; andthe imaging is performed by imaging the target site containing second,uncollapsed, GVPS type by applying imaging ultrasound to the targetsite, the imaging ultrasound applied at a pressure value lower than theacoustic collapse pressure value of the second gas vesicle structuretype.
 11. The ultrasound imaging method of claim 10, further comprising,after the imaging selectively collapsing the second gas vesiclestructure type by applying collapsing ultrasounds to the target site,the collapsing ultrasounds applied at a pressure value equal to orhigher than the selectable acoustic collapse pressure value of thesecond gas vesicle structure type.
 12. The ultrasound imaging method ofclaim 11, further comprising, after collapsing the second GVPS typeimaging the target site by applying imaging ultrasounds to the targetsite, whereby the sequence of visible images of the target site furtherincludes an image indicative of all uncollapsed GVPS types other thanthe first GVPS type and the second GVPS type.
 13. The ultrasound imagingmethod of claim 10, further comprising, before collapsing the first gasvesicle protein structure type imaging the target site by applyingultrasounds to the target site, the ultrasounds applied at a pressurevalue lower than the selectable acoustic collapse pressure value for thefirst gas vesicle structure type, thus obtaining an image indicative ofuncollapsed first and second gas vesicle protein structure types. 14.The ultrasound imaging method of claim 10, wherein the first GVPS typeand the second GVPS type are selected from GVPS types species ofAnabaena bacteria and Halobacterium bacteria.
 15. The ultrasound imagingmethod of claim 14, wherein the first GVPS type and the second GVPS typeare selected from GVPS types of Anabaena flos-aquae, Halobacteriumsalinarum, and Bacillus megaterium.
 16. The ultrasound imaging method ofclaim 10, wherein the first GVPS type comprises ΔGvpC and the secondGVPS type comprises ΔN&C, and the selectable acoustic collapse pressurevalue is 630 kPa.
 17. The ultrasound imaging method of claim 10, whereinthe acoustic collapse pressure value of the first gas vesicle proteinstructure type is selected from the first acoustic collapse pressureprofile at a value between 0.05% and 95% collapse.
 18. The ultrasoundimaging method of claim 17, wherein the acoustic collapse pressure valueof the first gas vesicle protein structure type is selected from thefirst acoustic collapse pressure profile at a value of 50% collapse. 19.The ultrasound imaging method of claim 10, wherein the acoustic collapsepressure value of a first gas vesicle protein structure type is selectedfrom the acoustic collapse pressure profile at a value that optimallymaximizes collapse of the first gas vesicle protein structure type whileminimizing collapse of the second gas vesicle protein structure type.20. The ultrasound imaging method of claim 19, wherein the optimallymaximizing collapse of the first GVPS type while minimizing collapse ofthe second GVPS type is determined by maximizing f1(p)-f2(p), whereinand ƒ1(p)=(1+e^((p-p) ^(c) ^()/Δp))⁻¹ and ƒ1(p)=(1+e^((p-p) ^(c)^()/Δp))⁻¹, and f1(p) and f2(p) correspond to an acoustic collapseprofile of the first GVPS and the second GVPS respectively.
 21. Theultrasound imaging method of claim 1, wherein the GVPS type comprises aplurality of gas vesicle protein structure (GVPS) types, each typeexhibiting i) an acoustic collapse pressure profile defined as acollapse function from which a collapse amount can be determined, andii) a selectable acoustic collapse pressure value, selectable acousticcollapse pressure values going from a lowest acoustic collapse pressurevalue to a highest acoustic collapse pressure value, wherein thecollapsing is performed by selectively collapsing GVPS type to acollapse amount higher than a collapse amount of each remaining GVPStype by applying collapsing ultrasound to the target site, thecollapsing ultrasound applied at a pressure value equal to or higherthan the selectable acoustic collapse pressure value of the GVPS typebeing collapsed and lower than an acoustic collapse pressure value ofsaid each remaining GVPS type or types; the imaging is performed byimaging the target site containing the remaining GVPS type or types byapplying imaging ultrasound to the target site, the imaging ultrasoundapplied at a pressure value lower than a lowest acoustic collapsepressure value of said each remaining GVPS type or types; and whereinthe method further comprises repeating the collapsing and the imaginguntil all GVPS types are collapsed, thus providing a sequence of visibleimages of the target site, the sequence being indicative ofimage-by-image decreasing remaining GVPS types.
 22. The acousticspectral method of claim 21, further comprising before a firstselectively collapsing step, imaging the target site by applying imagingultrasounds to the target site, the imaging ultrasounds applied at apressure value lower than the lowest selectable acoustic collapsepressure value; and after a last selective collapsing step, imaging thetarget site by applying further imaging ultrasounds to the target site,whereby the sequence of visible images of the target site furtherincludes an image indicative of all uncollapsed GVPS types and an imageindicative of an uncontrasted target site.
 23. The ultrasound imagingmethod of claim 21, wherein the imaging steps all occur at a samepressure, the same pressure being a pressure value lower than the lowestcollapsing acoustic collapse pressure value.
 24. The ultrasound imagingmethod of claim 21, further comprising an initial preparation step of:administering to the target site a contrast agent comprising theplurality of gas vesicle protein structure types.
 25. The ultrasoundimaging method of claim 21, wherein the GVPS types comprise acombination of GVPS types from species of Anabaena bacteria orHalobacterium bacteria.
 26. The ultrasound imaging method of claim 25,wherein the GVPS types comprise a combination of GVPS types fromAnabaena flos-aquae, Halobacterium salinarum and/or Bacillus megaterium.27. The ultrasound imaging method of claim 21, wherein the GVPS typescomprise ΔGvpC as a first GVPS type, ΔN&C as a second GVPS type, andGvpCWT as a third GVPS type, and a first selectable collapse pressurebeing 630 kPa, a second selectable collapse pressure being 790 kPa, anda third selectable collapse pressure being 1230 kPa.
 28. The ultrasoundimaging method of claim 1, wherein the GVPS comprises a GvpC variantcomprising a base GvpC protein having repetitions of a repeat regionflanked by an N-terminal region, and a C-terminal region, the GvpCvariant produced by at least one of: deleting at least one of theN-terminal region and C-terminal region; deleting 3 or more repeatedregions; deleting at least one repeated region immediately following theN-terminal region; adding a peptide to the C terminal or the N terminalregion; and substituting a sub-sequence within one or more repeatedregions.
 29. The ultrasound imaging method of claim 1, wherein the GVPSis a variant GVPS with GvpC removed.
 30. The ultrasound imaging methodof claim 10, wherein the first GVPS type and the second GVPS type areengineered to target different target sites.
 31. The ultrasound imagingmethod of claim 30, wherein engineering the first and second GVPS typesis performed by attaching a targeting moiety to the GVPS type.
 32. Theultrasound imaging method of claim 31, wherein the targeting moietycomprises a receptor-targeting peptide RGD.